1

CHAPTER-1

INTRODUCTION

1.1 CITALOPRAM

Citalopram (1) belongs to the class of antidepressant drug which is reuptake inhibitor of
selective serotonin. United State Food and Drug authority approved Citalopram (1) to
cure depression in 1998. It has licensed for depressive episodes, panic disorders and
obsessive-compulsive disorder in Australia, Denmark, Germany, Portugal, Poland, Spain,
United Kingdom and other European countries (Holmgren et al., 2004)

It is fourth in cost-effectiveness and fifth in efficiency in the ranking for ten

antidepressants made by National Institute for Health and Clinical Excellence on the
basis of meta- analysis. (Sprinks et al., 2002)

Citalopram

(1)
 2

1.1.1 History

Firstly, it was released by the trade name Cipramil in 1989 in Denmark. With the
increase in the reputation of drug, the drug could be recommended to any affected person
by the trade name Celexa, in 1998 in United State of America. It was first marketed in the
United State in 1998. (Benjamin &Paul, 2003) It made a profit of 720 million Denmark
Kroner in 1999 and became the best-selling product of Lund beck. When Lund beck‟s
drug patent ran out in 2003, it was the most dominant product of Lund beck.

1.1.2 Stereochemistry of Citalopram

Citalopram (1) consists of two enantiomers, which are different non-superimposable

mirror images of the same molecule that are R-citalopram (3) and S-citalopram (2).Their
individual characteristics have been studied by separating them. Escitalopram (2) is the S-
enantiomer of RS-citalopram (1). (Waugh et al., 2003) The mode of action of Citalopram
(1) is based on the affinity of S-enantiomer (2) for serotonin transporter that is double
than Citalopram‟s affinity and about thirty to forty times greater than R-enantiomer (3). It
results in the serotonin reuptake inhibition two times greater for Escitalopram (2) than
Citalopram and predicts that Citalopram‟s action is because of S-enantiomer (1). The
effect of Escitalopram (2) is superior to equal dose of Citalopram (1). This is illustrated
by many hypotheses. The pharmacokinetic studies have explained about the stability of S
and R enantiomers in vivo. The transformation of S-enantiomer (2) into R-enantiomer (3)
takes place, although this is not known why this occur less when Escitalopram (2) is
alone than when both enantiomers are there. (Davidson et al., 2004) It is interpreted that
R-enantiomer (3) not only counteracts the antidepressive action of Escitalopram (2) by
reducing its positive affects but also curtail the activity of Citalopram (1). This
counteraction was unexpected. (Davidson et al., 2004)
 3

S-Citalopram

(2)

R-Citalopram

(3)

1.2 METABOLISM OF ESCITALOPRAM

Almost 20% of the Escitalopram is metabolized by kidneys and remaining 80% by liver.
Escitalopram (2) is metabolized in the liver to slight lipid loving compounds which are
immediately eliminated by urine. (Waugh et al., 2003; Sogaard et al., 2005; Louis, 2006)
Oxidative metabolism of Escitalopram (2) to S-desmethylcitalopram (4) and S-
didesmethylcitalopram (5) takes place through N-desmethylation. (Sidhu et al., 1997;
Rochat et al., 1996; Oyehaug et al., 1984; Rochat et al., 1995)

N-desmethylation of Escitalopram (2) to S- desmethylcitalopram (4) is carried out by

three liver cytochrome P450 enzymes that are cytochrome P3A4, cytochrome P2C19 and,
to a lesser extent cytochrome P2D6. (Von Moltke et al., 2001) Cytochrome P2D6
mediates the desmethylation of S-desmethylcitalopram (4) to S-didesmethylcitalopram
 4

(5). (Von Moltke et al., 2001) Contray to this, metabolism may be mediated by aldehyde
oxidase and monoamine oxidase A and B (MAO-A and MAO-B) in brain. (Rochat et al.,
1998; Kosel et al., 2002) It has been observed that Escitalopram‟s metabolites possess
one-seventh of the capability of Escitalopram (2) for inhibiting reuptake of serotonin.
(Hyttel et al., 1992; Sugars et al., 2005)

Scheme: 1.1 Metabolism of Citalopram

1.2.1 Metabolites of Escitalopram

There are two metabolites of Escitalopram (2). One is N-desmethylescitalopram (4) and
other is N, N-Didesmethylescitalopram (5). The action of both metabolites is unimportant
than total action of Citalopram. (Von Moltke et al., 2001)

1.2.1.0 N-Desmethylescitalopram

Desmethylescitalopram (4) is an active metabolite of antidepressant drugs Citalopram

(enantiomers) (1) and Escitalopram (S-enantiomer of Citalopram) (2). Escitalopram
oxalate is used to synthesize and characterize N-Desmethylescitalopram (4) by a one-step
N-Desmethylation reaction. The synthesized impurity N-Desmethylescitalopram (4) is
 5

purified by column chromatography. The purified impurity is analyzed by HPLC. The

functional group in the compound is analyzed using FT-IR and its molecular weight is
determined by LC-MS. The structure of the purified compound is determined by 1HNMR
spectroscopy and 13CNMR spectroscopy analysis respectively.

Like Citalopram (1) and Escitalopram, (2) Desmethylcitalopram (4) also functions as
selective serotonin reuptake inhibitor (SSRI).

N-Desmethylescitalopram

(4)

1.2.1.1 N, N-Didesmethylescitalopram

Didesmethylescitalopram (5) is an active metabolite of antidepressant drugs Citalopram

(enantiomers) and Escitalopram (S-enantiomer of Citalopram) (2). Escitalopram oxalate
is also used to synthesize and characterize N-Didesmethylescitalopram (4). First of all N-
Desmethylation of Escitalpram (2) occurs followed by N, N-Didesmethylation (5) of
Escitalopram (1). The synthesized impurity N, N-Didesmethylescitalopram (5) is purified
by column chromatography. The purified impurity is analyzed by HPLC. The functional
group in compound is analyzed by using FT-IR and its molecular weight is determined by
LC-MS. The structure of the purified compound is determined by 1HNMR spectroscopy
13
and CNMR spectroscopy analysis respectively. Like Citalopram (1) and Escitalopram
(2), Didesmethylescitalopram (5) also functions as reuptake inhibitor of selective
serotonin.
 6

N, N-Didesmethylescitalopram

(5)

1.3 Selective Serotonin Reuptake Inhibitors (SSRIs)

In the modern era, antidepressant medications which are reuptake inhibitors of specific
serotonin serve as a primary decision to cure direct to extreme depressive disease,
different fears and individual issues. These work by increasing the activity of serotonin
by stopping its reuptake at presynaptic terminals. (Updated 2013). (Gartlehner et al.,
2008)

Citalopram is such a reuptake inhibitor of serotonin that needs dosage adjustment in old
people. (Pollock et al., 2009)

1.3.1 Types of Selective Serotonin Reuptake Inhibitors

At present, there are seven SSRIs recommended in United Kingdom.

 Citalopram (Celexa) (1)

 Escitalopram (Lexapro) (2)
 Fluoxetine (Prozac)
 Paroxetine (Paxil, Pexeva)
 Sertraline (Zoloft)
 Vilazodone (Viibryd)
 7

1.3.2 Mechanism of Action of Selective Serotonin Reuptake Inhibitor

It works by boosting up serotonin (a neurotransmitter) levels in brain. Mood, emotions

and sleep are influenced by it. Serotonin is generally reabsorbed which is also called as
its reuptake by nerve cells after transmission of nerve impulse. SSRIs work by inhibiting
reuptake which means more serotonin is assessable to transmit additional messages
between close-by nerve cells. Depression and related mental health conditions results by
low serotonin level. On the other hand increase in serotonin levels can better symptoms
and make individuals more receptive to various kinds of treatment. (Figure1.1). (Wade et
al., 2002)

(A) Hypothetical serotonergic neuron.

(B) Short-term administration of SSRI hinder reuptake of serotonin at cell body

prompting decreased rate of nerve impulse transmission by neuron. This is just because
of activity of serotonin on 5HT1A auto receptors.

(C) Long-term administration results in down regulation of 5HT1A auto receptors,

prompting increased rate of nerve impulse transmission by neuron. There is too much
serotonin which acts post synaptically, when reuptake of serotonin is inhibited. (Norman,
1999).

Figure: 1.1 Mechanism of Action of Selective Serotonin Re Uptake Inhibitor

 8

1.3.3 Relation of Selective Serotonin Reuptake Inhibitor to Citalopram

Sufferers of depression possess „chemical imbalance‟ in their brain. There is shortage of

adequate quantity of neurotransmitter serotonin which holds significant role to play in
behavior, mood and sleep. Reuptake channels on the surface of neurons are blocked by
Citalopram. Serotonin gets higher exposure at serotonin receptors because much of the
time is required to dislodge serotonin from synaptic cleft. There is much time for
serotonin to bind to receptor, thus gives the impact of having an elevated accumulation of
neurotransmitter in synaptic cleft. An elevated response occurs due to strong interactions
between neurotransmitter and receptor. Citalopram is classified as particular serotonin
reuptake inhibitors because of this mode of action.

Figure: 1.2 Role of Citalopram as Selective Serotonin Reuptake Inhibitors

1.3.3.1 Up Regulation

In case if there is depletion of neurotransmitter acting at a postsynaptic receptor, it results

in a redeeming increment in quantity of receptor sites on postsynaptic cell surface. This is
termed as up regulation.
 9

Figure: 1.3 Up regulation

1.3.3.2 Down Regulation

In case if there is dependable increment in accessibility of neurotransmitter at a synaptic

receptor site, it results in a decline in quantity of receptors on cell surface. This is termed
as down regulation.

Figure 1.4 Down regulation

1.4 MEDICAL USES

Food and drug authority has suggested Escitalopram (2) for cure of depressive disorders
in young people and anxiety disorders in young people and grown-ups. It is suggested for
Depression (MDD) and anxiety issues in European nations and Australia.

 General anxiety disorder (GAD)

 Obsessive-compulsive disorder (OCD)

 Panic disorder with or without agoraphobia

 Social anxiety disorder (SAD)

1.4.1 Depression

Indications of depression are:

 10

 Feeling pessimistic and fearful

 Feeling valueless, powerless or hopeless

 Loss of eagerness and joy in daily routine

 Low spirit, danger loving and thinking of suicide

 Psychomotor hindrance

 More or less sleep than regular

 More or less eat than regular

For the treatment of primary depressive disorders, Escitalopram (2) was suggested by
administrative authorities. An efficient survey and system meta-investigation looking at
the viability and adequacy of 21 antidepressant drugs in 2018 indicated escitalopram (2)
to be a standout amongst the best antidepressants. Proof for adequacy of citalopram (1)
for treating depression in kids is unverifiable. (Cohen, 2007; Carandang et al., 2011)

1.4.2 Anxiety Disorders

When a person faces too much anxiety and stress for about 6 months, then these anxiety
disorders occur. Symptoms are;

 Insomnia

 Weakness

 Inattention

 Irascibility

 Muscle stiffness

 Sleep unsettling influence

Escitalopram (2) appears to be effectual in treating anxiety disorders. (Bech et al., 2010)
 11

1.4.3 Panic Disorder

For treatment of anxiety attacks, Citalopram (1) is accredited in the United Kingdom
(U.K) and other European countries. Initially dosage is 10mg/d for a week, and then
increased to 20-30mg/d.

1.5 SIDE EFFECTS

The vast majorities of the side effects of SSRIs are identified with dosage and may be due
to serotonergic impacts. The reason behind this is that many neurotransmitters are
available to interact with receptors such as dopaminergic, cholinergic, histaminic and
subtype receptors when serotonin reuptake inhibition is increased. (Nelson,
1997) Nausea (mostly results from stimulation of 5-HT3 receptors and can be lessened by
decreasing dose of SSRI) and Gastrointestinal (GI) disturbances are most common side
effects of SSRIs. (Goldstein and Goodnick 1998; Spigset, 1999) Sexual dysfunction,
sleep disturbance and weight gain are most detrimental side effects of SSRIs. Citalopram
is best-tolerated SSRI.

1.5.1 Sexual Dysfunction

Those patients who had experienced sexual dysfunction with other SSRI are not being
affected by Citalopram (1). Most common side effect of Citalopram (1) therapy is
Anorgasmia. (Noble & Benfield, 1997) Research reported the frequency of sexual
dysfunction in patients treated with Citalopram compared to those treated with other
SSRIs. (Rosen et al., 1999)

1.5.2 Weight Gain

During premarketing clinical trials of SSRIs, weight gain was unusually reported.
However, data studies depicted that citalopram (1) rarely causes weight gain. (Mackle &
Kocsis, 1998) According to a clinical research, out of the series of 18 patients, 8 were
 12

reported with mood and anxiety disorders. After acquiring citalopram for 35 days, they
had an average weight gain of 15.71b (7.1). (Bouwer & Harvey, 1996)

1.6 CHOLINESTERASE

It is an esterase (hydrolase enzyme) that catalyzes the breakdown of choline-based esters,

many of which behave as neurotransmitter. Two types of enzyme belong to this; one is
acetylcholinesterase (erythrocyte cholinesterase) which is primarily found in chemical
synapses and red blood cells membranes. Second one is butyrylcholinesterase, which is
present primarily in blood plasma. That is why butyrylcholinesterase is also called as
plasma cholinesterase.

1.6.0 Acetylcholinesterase

It catalyzes the degradation of neurotransmitters such as acetylcholine and some other

choline esters. About 25000 molecules of acetylcholine are cleaved per second by each
molecule of acetylcholinesterase. (Quinn, 1987; Taylor & Radić 1994)

1.6.1 Subsites of Acetylcholine

There are two subsites (anionic subsite and esteratic subsite) of the active site of
acetylcholinesterase. (Sussmann et. al., 1991; Sussmann et. al., 1993) The positive
quaternary amine of acetylcholine, other cationic substrates and inhibitors are
accommodated by the anionic subsite. Cleavage of acetylcholine to choline and acetate
occurs at the esteratic subsite that comprises a traid of three amino acid. Presynaptic
neuron release acetylcholine into synaptic cleft during neurotransmission. Acetylcholine
receptors on post-synaptic neuron bind it relaying the signal from the nerve. Signal
transmission is ceased when acetylcholinesterase located on the post-synaptic membrane
hydrolyses acetylcholine. Acetylcholine is synthesized by reacting with acetyl-CoA
through the action of acetylcholine transferase when released choline is again taken up by
pre-synaptic neuron. (Figure 1.5). (Whittaker, 1990; Purves et al., 2008)
 13

Figure: 1.5 AChE Mechanism of action

1.6.2 Butyrylcholinesterase (BuChE)

Acetylcholine and various other esters are hydrolyzed by an enzyme

butyrylcholinesterase but have no known physiological function. It is also called as
pseudo cholinesterase or non-specific cholinesterase. (Chatonnet et al., 1998)

1.6.3 Mechanism of Action of AChE and BuChE in the Cholinergic

Synapse

Initially, during neurotransmission acetylcholine containing vesicles come in contact with

the presynaptic membrane, this fusion results in the release of acetylcholine into synaptic
cleft. (Figure 1.6). Then interaction of acetylcholine with cholinergic receptors on the
post synaptic neuron occurs by diffusing across synaptic cleft. Acetylcholine is
hydrolyzed to acetate and choline by Acetylcholinesterase which is a serine hydrolase
and necessary for maintaining pulsatic cholinergic stimulation. The released choline is
used for synthesis of acetylcholine as it is carried back to presynaptic neuron. (Adam,
1992)

According to a hypothesis, this acetylcholine can also be hydrolyzed to acetate and

choline by glial butyrylcholinesterase which is known as primary extra synaptic
acetylcholine hydrolyzing enzyme. (Giacobini, 2001) Choline is then returned to the
 14

synaptic cleft for reuptake into cholinergic neurons. (Mesulam et al., 2002; Daikhin &
Yudkoff, 2000) Acetylcholine activity is also observed in cerebrospinal fluid and
extracellular fluid along with its activity at neuronal synaptic cleft. (Yamada et al., 1996)

Figure: 1.6 Hydrolysis of ACh by AChE and BuChE

1.6.4 Citalopram and Cholinesterase Inhibitors in Alzheimer Disease

One of the central nervous system‟s degenerative disorders is Alzheimer‟s disease (AD)
with progressive cognitive, psychological, functional and behavioral decline. Agitation,
depression (Drye et al., 2011) and language deficit (Nakamura et al., 2014) are common
among (Olin et al., 2002; Porsteinsson et al., 2014) elderly with AD and depression is
considered as comorbidity (Blazer et al., 1997) Depressive symptoms in elderly or
patients with AD appear at 7%-42%. (Bruce et al., 1994) Dementia in combination with
depression is strongly associated with an increase of cognitive dysfunction. (Porsteinsson
et al., 2008) A case study in 2002 (Lyketsos et al., 2000) found that 20% of the persons
who suffered from dysphoria also suffered from irritability and many kinds of depression.
(Montgomery & Djarv, 1996) Furthermore, due to its long-term nature, the illness
requires psychopharmacological treatment over long periods of time. However, patients
with depression can be prescribed with higher doses of citalopram (1). Medication like
rivastigmine and citalopram (1) are used in everyday clinical praxis. Rivastigmine is an
Acetyl cholinesterase Inhibitor (AChEI) with the activity of a Butyrylcholinesterase
 15

Inhibitor (BuChEI) which showed a better cognitive progression in patients with AD.
(Darreh-Shori et al., 2002) Both these enzymes cease the role of acetylcholine in synaptic
cleft by breaking it to acetate and choline. Citalopram (1) inhibits inactivated and
substrate-activated forms of BuChE. (Walsh et al., 2011; Rockwood et al., 2011)

Citalopram (1) is used to treat depressive symptoms that are very common in patients
with AD. A research study summarized and showed that citalopram (1) was effective as
an antidepressants therapy. (Keller, 2000)

A study of Leblhuber F. (1994) showed that there was an improvement in 20 patients

with dementia regarding confusion, restlessness, and behavioral disturbance symptoms
after they were treated with citalopram (1) for three weeks. It also emphasized the effect
of citalopram (1) as an emotional “stabilizer” for those patients, (Leblhuber, 2014) in
other study investigators of a Nordic multicenter; found that Alzheimer‟s patients who
had been treated with citalopram (1) showed greater improvements for considering
irritability and restlessness. (Nyth & Gottfries, 1990) Citalopram (1) is used as an
antidepressant medicine because it is less expensive and clinically safe; tolerability and
efficacy are guaranteed, and it does not cause adverse cardiovascular or anticholinergic
effects.(Parker & Brown, 2000) In comparison to citalopram (1), other anti-depressant
drugs like; mirtazapine, venlafaxine and reboxetine, or non-conventional ones like
hypericum, citalopram (1) have shown to be significantly less effective than escitalopram
(2) but more effective than paroxetine and reboxetine. Therefore, citalopram (1) is more
reliable as an antidepressant therapy for the AD patients and thus more acceptable.
Citalopram (1) is implicated in cell survival and it does not interfere with the molecular
controls of the cell. (Cipriani et al., 2012)

1.7 DESMETHYLATION OF CITALOPRAM BY

CHLOROETHYL CHLOROFORMATE

Citalopram can also be converted into desmethylescitalopram by using Chloroethyl

chloroformate by following method. In the presented study N-desmethylation is carried
by using Iodine, but given mechanism is another method of desmethylation of citalopram
 16

Figure: 1.7 Desmethylation by Chloroethylchloroformate

1.8 OBJECTIVES OF STUDY

The objectives of the study were to

 Synthesize N, N-Didesmethylescitalopram (5)

 Synthesize analogues of N,N-Didesmethylescitalopram (5)

 Cholinesterase biological evaluation of N,N-Didesmethylescitalopram (5) and its

analogues (36 and 37)

1.9 SIGNIFICANCE OF STUDY

Didesmethylescitalopram is the metabolite of antidepressant drug Citalopram.

Citalopram shows effective behavior in treating depression. Didesmethylescitalopram
plays same role in depression treatment as Citalopram. Citalopram and its metabolites
show cholinesterase inhibitor activity. Further analogues of Didesmethylescitalopram (36
& 37) are prepared to check their relative behavior to inhibit activity of cholinesterase
with respect to Citalopram.
 17

CHAPTER-2

REVIEW OF LITERATURE

Citalopram (1) is known to be an antidepressant which is reuptake inhibitor of serotonin

and is commonly worldwide. R-(3) and S-(2) are its two enantiomeric forms. The S-
enantiomer is also known as Escitalopram (2). Citalopram (1) is metabolized to N-
Desmethyl citalopram (4) and N, N-Didesmethyl citalopram (5) in liver by enzymes.
From literature survey, it is concluded that both these metabolites function as selective
serotonin reuptake inhibitors. Citalopram (1) analogues act as unique probes for S1 and
S2 binding sites of serotonin transporter. The possible use of these metabolites can be of
great benefit in the field of medicine. My target is one of the metabolite of Citalopram (1)
i.e; N, N-Didesmethyl citalopram (5).

For testing the inhibitory potential of 5-(aroylhydrazinocarbonyl) escitalopram against

cholinesterases, its unique series have been outlined and synthesized. The vigorous
compound of the series was 3-Chlorobenzoyl- (6) with IC50 1.80 ± 0.11mM for
inhibition of acetylcholinesterase and the most effective compound was 2-bromobenzoyl-
(7) with IC50 2.11 ± 0.31mM for inhibition of butyrylcholinesterase (BChE). Enzyme
inhibition was influenced by moderate electron donating groups (increase), electron
withdrawing groups (decrease) and size or position of the substituents. In rendezvous
study of acetylcholinesterase and butyrylcholinesterase, the ligands (6) and (7)
demonstrated the scores of 5873 (ACE value 64.91kcal/mol), and 5665 (ACE value
140.15kcal/mol) for acetylcholinesterase likewise, 6015(170.89kcal/mol) and 6150(ACE
 18

256.84kcal/mol) for butyrylcholinesterase. While ligand (8) showed scores of 5754 (ACE
value 203.14) for acetylcholinesterase and ligand (9) outlined 5993 (235.95kcal/mol) for
butyrylcholinesterase. (Mehr-un-Nisa et al., 2017)

R=3-chlorobenzoyl-(6)
R=2-bromobenzoyl-(7)
R=4-chlorobenzoyl-(8)
R=4-iodobenzoyl-(9)

The current research work described here covers a modified access to prepare the
enantiopure Escitalopram (2), extensively used anti-depressants by involving four-step
process viz., (i) desmethyl reaction of Citalopram/bromoescitalopram (10). (ii)
Desolation of desmethylcitalopram /bromo desmethyl escitalopram (11), using Di-p-
toluoyl-D-Tartaric acid (DPTTA) in methanol, which is consequently given unique key
intermediates to ultimately give Escitalopram (2) by means of diastereomeric salt
formation and further, optimized the unique resolution condition and other key defectors.
(iii) Desolation of Desmethyl Escitalopram (4)/ Bromodesmethylescitalopram (11). (iv)
This step applies to enantiomerically pure desmethyl escitalopram(4), on alkylation with
formaldehyde give effective pharmaceutical element1 with valuable yield (75- 78%) and
constitution.(v) One more enantiomerically pure Bromodesmethyl escitalopram(11), on
alkylation with formaldehyde and formic acid, followed by cyanation affords 1 with
valuable yield (~70%) and constitution. In the current research study reports the
commercially beneficial new synthetic process of Escitalopram (2) by the unique
desolation of desmethylcitalopram/ bromo desmethylescitalopram (11) with valuable
yield and constitution of product. (Bobbali et al., 2016)
 19

(10)

(11)

Chemical process associates the synthesis of a single product or certain products by

mixing compounds at certain proportions and conditions. In the current study,
Escitalopram oxalate is used to prepare and characterize N-desmethyl escitalopram (4) by
a one-step N- desmethylation reaction. The prepared impurity N-desmethyl escitalopram
(4) was filtered by column chromatography. The filtered impurity was analyzed by
HPLC. The functional group in the compound was analyzed using FT-IR and its
molecular weight was resoluted by LC-MS. The structure of the filtered compound was
determined by 1HNMR spectroscopy and 13
CNMR spectroscopy analysis respectively.
(Saranya et al., 2015)

For testing the effectiveness of serotonin reuptake inhibitors bifunctional ligand at SSRIs
receptors and opioid bifunctional ligands at μ-, δ- and  opioid receptors, a unique series
of these ligands have been designed. Low partiality was exhibited by many compounds
for SSRIs and  opioid receptors while high partiality for μ- and δ-opioid receptors.
Maximum binding profiles for 1-opoid receptor binding site have been shown by ligands
Dmt-D-Nli-Gly-Phe (4-F)-(12) with DGbind (12.9 kcal/mol) and Ki value (1.01 nM) and
Dmt-D-Tic-Phe (4-F)-(13) with DGbind (12.39 kcal/mol) and Ki value (0.40 nM).
 20

Ligand Tyr-D-Ala-Gly-Phe-(14) showed both abilities, the reuptake inhibition of

serotonin (antidepressant action) and opioid activities. That is why it could be used to
cure depression. (Mehr-un-Nisa et al., 2015)

To enact the inhibitory potential of triazoles and tetrazole (15) regarding

butyrylcholinesterase and acetylcholinesterase, a unique sequence of these compounds
have been prepared and administered to research. Highest acetylcholinesterase inhibition
was shown by 4-chlorophenyl- (16) and 2-methylphenyl- (17) escitalopram triazole
derivatives whereas good butyrylcholinesterase inhibition by 2-fluorophenyl- (18) and 4-
fluorophenyl (19). It was noticed that electron donating groups boost the inhibition
whereas it was found to decrease by electron withdrawing groups. (Mehr-un-Nisa et al.,
2015)

R=4-chlorophenyl triazole-(16)
R=2-methylphenyl triazole-(17)
R=2-flurophenyl triazole-(18)
R=4-fluorophenyl triazole-(19)
 21

(15)

Aniline was substituted on 5-position of dihydroisobenzofuran ring of Citalopram (1)

using an ethylamino linker to get rhodamine fluorophore. The uptake of [3H] 5-HT
uptake in COS-7 cells was inhibited by resulting unique radiant rhodamine labeled ligand
(20) with comparative efficacy to the tropane-based JHC 1-064 (21). Confocal
microscopy manifested the anticipation of Serotonin transporter with compound (20) in
HEK293 cells. (Zhang et al., 2013)

(20)

(21)
 22

An underlying target for antidepressant medications is serotonin transporter. Analogues

of Citalopram (1) were prepared by modifying N, 4, 5, and 4′ positions of it. These
analogues endured the steric bulk at primary orthosteric binding site (S1) of serotonin
transporter along with dimeric ligands (22 and 23). These dimeric compounds had
comparable affinities for serotonin transporter (S1) site. Both dimeric compounds had
comparable affinities for the SERT S1 site. However, the N-substituted analogue (23)
actively regulated the binding of [3H] S-1 via low affinity binding site (S2). (Banala et al.,
2013)

(22)

(23)

The intent of the current study is to the preparation, characterization and dissolution
studies of inclusion complexes of escitalopram oxalate. Inclusion complex of drug was
synthesized by using various cyclodextrins such as ßCD and HPßCD in distinctive ratios
from (1:1 to 1:2) by using kneading method. Phase solubility studied classified as AL
type marked an increase in solubility of drug with arise in concentrations of
cyclodextrins. Fourier transform infrared spectroscopic studies demonstrated that there
 23

was an absence of well-defined drug cyclodextrin interaction and X ray diffraction

studies certified that the overall crystalline peaks of complexes were decreased as
compared to pure drug. All inclusion complexes and physical mixture showed high
dissolution rate as compared to pure drug. The dissolution rate elevates with increasing
the ratio of HPßCD (100.5 ± 2.64% drug release KN6 at 60 mins) as compare to ßCD
(81.5 ± 2.32% drug release KN3 at 60 mins). Also, the highest 81.7±2.66 % drug release
noted at 30 mins for HPßCD using kneading method. Certainly, it was concluded that
inclusion complex synthesized with kneading method HPßCD (KN6) and ßCD (KN3)
showed sophisticated dissolution enhancement capacity than physical mixing and pure
drug in 1:2 drug to cyclodextrin ratio. (Purohit et al., 2012)

The compound (24) and (25) were prepared in a Br/125I exchange reaction as two unique
radioligands for serotonin transporter. Using these ligands, experiments were performed
on rats. 0.34% of injected dose had concentrated in brain after one hour endovenous
injection of 24. The value approached to 2.4 after 2 hours leading to highest
hypothalamus to cerebrum ratio.

Compound (26) was inspected in a pig single photon emission computed tomography
(SPECT) study. After IV injection, hypothalamus to cerebrum ratio was 1.2 between 1 -2
hours.

The whole research showed that Citalopram and Escitalopram‟s iodine labeled
derivatives are not preferable to any other SPECT tracer for serotonin transporter, such as
[123I]. (Madsen et al., 2011)

(24)
 24

(25)

I=123I (26)

To cure anxiety and depression clinically, (±)-Citalopram (1) and its enantiomer,
escitalopram (2) are used. A list of (±)-4- and 5-substituted citalopram (1) analogues were
designed, prepared and estimated for binding at the serotonin transporter in rodent tissue.
High serotonin transporter binding affinities were demonstrated by these analogues. For
binding at homologous bacterial Leucine transporter, enantiomeric pairs of (1) and
(27) were observed

R=5 bromo citalopram-27

Refined and active formation of N-desmethylcitalopram (4) and N, N-

didesmethylcitalopram (5) is given. 1-chloroethyl chloroformate is used for N-
desmethylation of Citalopram to give 89% yield. Alkylation of (28) with 1-(3-
bromopropyl)-2, 2, 5, 5-tetramrthyl-1-aza-2, 5-disilacyclopentane (29) results in the
complete formation of (5). (Jin et al., 2007)
 25

(28)

(29)

The current innovation defines a modified mechanism for the synthesis of highly pure
Citalopram (1) and its bromide salt, also called as Citalopram hydrobromide (most
familiar antidepressant medication). Further aspects of innovation are:

 Crystalline Bromiodol is desolated.

 Desmethylcitalopram (4) which is formed during the cyanide exchange reaction is
transformed to Citalopram (1) by heating with an amalgam of formaldehyde and
formic acid in chloroform.
 Extraction morphology is used to purify derived Citalopram (1). (Siddique et al.,
2005)

The three C-11 labelled tracers S-[N-methyl-C-11] citalopram ([C-11]-(30), S-[N-methyl-

d3-C-11] citalopram ([C-11]-(31), and S-[N-methyl-C-11] citalopram-α,α-d2 ([C-11]-(32)
were prepared and injected in rats. Circulation of radioactivity was observed in rats.
Escitalopram (2) was used in multi-step synthesis of deuterated analogue of (S)-
desmethylcitalopram (4), (S)-1-(4-fluorophenyl)-1-(3-methylamino-[3-d2]-propyl)-1,3-di-
hydro-isobenzofuran-5-carbonitrile (33). (Madsen et al., 2004)
 26

R=H, X=H-(20)
R=D, X=H-(31)
R=H, X=D-(32)

(33)

Citalopram (1) is frequently used drug in Swedish forensic postmortems. High

performance liquid chromatography was used for enantioselective analysis of Citalopram
and its metabolites desmethylcitalopram (4) and didesmethylcitalopram (5) in femoral
blood from 53 postmortems. With increasing the concentration of Citalopram (1), high
S/R ratios were observed.

Cytochrome P450, Cytochrome P3A4 and Cytochrome P2D6 are used to metabolize
Citalopram (1). Only two poor metabolizers respecting polymorphic Cytochrome P2D6
and no poor metabolizers respecting polymorphic Cytochrome P2C19 were exposed.
Such pharmacokinetic interactions are a more significant dilemma than metabolic
deficiency. This has been proposed by ubiquity of drugs that are metabolized by these
enzymes or inhibit these enzymes. (Holmgren et al., 2004)
 27

In Swedish vigorous volunteers, the pharmacokinetics of enantiomers of Citalopram,

desmethylcitalopram and didesmethylcitalopram respecting Cytochrome P2C19 and
Cytochrome PY2D6 geno and phenotypes have been observed. Representatives with
variant genotype and phenotype were treated with Citalopram for 7 days. After the 12
hours of last dosage of Citalopram, all urine was collected. In PM of mephenytoin, AUC
of S-(2) was found to be fundamentally higher but not AUC of R-(3) as compared to
Ems. Minor dosage of Citalopram is demanded by PMs. (Herrlin et al., 2003)

Three inhibitors of serotonin reuptake were prepared by retrieving the 5-nitrile group in
Citalopram with methyl, acetyl and piperidinyl. The compound (34) was prepared in 70-
89% yield by employing [C-11] methyl iodide in a Stille reaction. Similarly, by
employing [C-11] carbon monoxide in palladium mediated reaction the compound (35)
was prepared in 65% chemical yield. The activation of corresponding trimethyltin and
tributyltin precursor was an important factor in the activity of [C-11]-34. The acceptable
blood-brain barrier penetration was presented by [C-11]-34 but [C-11]-35 did not show
this in rodent research. In accordance with the known distribution of serotonin
transporters, there was an aggregation of radiotracers in brain. After pretreatment with
Citalopram, unique binding was slightly impeded. Hence, no pertinent properties as
radioligand for determination of serotonin transporter are shown by [C-11]-34. (Madsen
et al., 2003)

(34)
 28

(35)

There is minor pharmacokinetic, efficacy or tolerability research on adolescents; though

the prescribing of selective serotonin reuptake inhibitors is comprehensive for them. In
fact, two analyses (retrospective and prospective) were observed in adolescents treated
with Citalopram. This research briefs all the pharmacokinetic data. Serum concentrations
of Citalopram, desmethyl citalopram and didesmethyl citalopram in adolescents
respecting daily dosage were explained by this research. Patients less than 21 years were
analyzed.

Foremost outcomes were;

 Marked inter individual fluctuation of serum (1), (4), and (5) concentrations in all
recommended doses.
 Within the dose range of 19-59mg/day, there was an identical conversion of (1) to
(4) and of (4) to (5).
 Girls showed fundamentally greater values on analyzing dose corrected
concentrations of (1) and (4) than boys.

In short, current research temporarily supports impact of sex. (Reis et al., 2002)

In human hepatic microsomes and expressed cytochromes, the conversion of

escitalopram (2) to desmethyl escitalopram (4), and (4) to didesmethyl escitalopram was
observed (5). The conversion of R-Citalopram (3) was also discussed. Cytochrome
P2C19, Cytochrome P2D6 and Cytochrome P3A4 were used to transform (2) to (4).
Contributions to net intrinsic clearance were 38%, 36% and 27% for Cytochrome P2C19,
Cytochrome P3A4 and Cytochrome P2D6 respectively. Only CYP2D6 mediates the
formation of (5) from (4). The formation of (4) was lessened to 62% and 83% by
 29

ketoconazole and quinidine respectively. Cytochrome P2D6 was weakly inhibited by (2)
and (4). Drug interactions and genetic polymorphism rarely affect Escitalopram. (Von
Moltke et al., 2001)

According to this earlier research, human cytochromes (Cytochrome P2C19, Cytochrome

P2D6 and Cytochrome P3A4) and hepatic microsomes mediate the conversion of racemic
Citalopram to desmethylcitalopram. (Kobayashi et al., 1997; Rochat et al., 1997; von
Moltke et al., 1999b). Studies reveal contribution of 37%, 28% and 35% for Cytochrome
P2C19, Cytochrome P2D6 and Cytochrome P3A4 respectively to this reaction at low
concentrations of Citalopram. The Escitalopram (2) and desmethyl escitalopram (4) than
their corresponding R-isomers possess higher antidepressant action. (Hyttel et al., 1992)

Biotransformation of R- and S-enantiomers of Citalopram to desmethylcitalopram and of

R- and S-desmethylcitalopram to didesmethylcitalopram mediated by human hepatic
microsomes and cytochromes and indicated by complementary DNA-transfected human
lymphoidblastoid cells have been assayed by current research. The human cytochromes
P450 impeding capability of Citalopram and desmethylcitalopram were also studied.
Cytochrome P2C19, Cytochrome P2D6 and Cytochrome P3A4 are used for
biotransformation of Citalopram (1) to desmethyl escitalopram. Insignificant inhibition of
Cytochrome 2C9, cytochrome 2E1 and cytochrome 3A and very weak inhibition of
cytochrome 1A2, cytochrome 2C19 and cytochrome 2D6 were caused by Citalopram and
desmethylcitalopram. (VonMoltke et al., 1999)

Citalopram (1) is such an antidepressive medicine that is reuptake inhibitor of selective

serotonin. It is racemic in nature, though it‟s mode of action remains primarily in S-
enantiomer (2). Cytochrome P450 is used for mediating the metabolism of Citalopram (1)
to N-desmethylcitalopram and N, N-didesmethylcitalopram. By the deamination of (1),
(4) and (5), another inactive metabolite of (1) which is Citalopram propionic acid is
prepared. Objective of current research was to observe the synthesis of Citalopram
propionic acid enantiomers from (1), (4) and (5). It was demonstrated that Citalopram
propionic acid was NADP-independent by incubation of racemic citalopram, racemic
desmethylcitalopram and racemic didesmethylcitalopram in presence of NADP. For
 30

synthesis of Citalopram propionic acid from citalopram, desmethylcitalopram and

didesmethylcitalopram, monoamine oxidases and aldehyde oxidase were also analyzed.
Both monoamine oxidase inhibitor of type A and B inhibited Citalopram propionic acid
formation. In short, deamination possess a lot of significance in biotransformation of (1),
(4) and (5) peculiarly in brain. (Rochat et al., 1998)

Patients (10 in numbers) with mean age 77 years going through mental illness and serious
psychotic disorders in took Citalopram for 4 days (11mg/day) and then dosage was
increased to 22mg/day for further 14 days. Fundamental improvement in behavior was
shown by 7 patients after 18 days of treatment. Enantiomeric plasma levels of Citalopram
and desmethylcitalopram were also measured using Ultraviolet detection. For
Escitalopram and inactive R-Citalopram, plasma level ranges were 11.3-91.3ng/ml and
12.5-94.5ng/ml respectively. Similarly, it ranged from 10-20ng/ml for S-
desmethylcitalopram and 9-20ng/ml for R-desmethylcitalopram. Plasma level to dose
ratio was depicted to be 1.89 by Overo in 1981 for 50 young patients. While according to
this current research it comes out to be 3.49 for older patients implying variations in
cytochrome P2C19 are linked to age. (Foglia et al., 1997)

For analyzing primary Citalopram‟s enantiomers and its metabolites i.e;

desmethylcitalopram and didesmethylcitalopram in plasma, a high performance liquid
chromatography technique has been developed using fluorescence detection. The
derivatives of Citalopram N-oxide and Citalopram propionic acid are separated by this
method. In 25 patients treated with different quantity of Citalopram ranging from 25-
75mg/day, plasma levels of S- and R- Citalopram, desmethylcitalopram and
didesmethylcitalopram were measured. Fundamental correlations in plasma levels were
observed between S-Citalopram and R-Citalopram and between S-desmethylcitalopram
and R-Citalopram. Enantiomeric ratios were influenced by co-medications very slightly.
(Rochat et al., 1995)

Positron Emission Tomography was used to observe non obstructive in vivo studies of
sites in human brain which are for serotonin uptake by labeling Citalopram (1) with C-11.
Approximately, seventeen minutes were taken for accomplishment of preparation. [C-11]
 31

Methyl iodide was used as a precursor. Purification, characterization and preparation of

novel efficacy are explained. (Hayden et al., 1990)
 32

CHAPTER – 3

RESEARCH METHODOLOGY

This research work was performed in the laboratories of Department of Chemistry,

Division of Science and Technology, University of Education Township Campus Lahore.

3.1 MATERIAL

3.1.1 Apparatus

 Alumimium foil

 Appendrophs

 Beaker

 Filter paper

 Hot plate

 Iron stand

 Magnetic bar

 Measuring cylinder
 33

 pH paper

 Pipes

 Pipette

 Quick fit flask

 Reflux condenser

 Round bottom flask

 Separating funnel

 Spatula

 Stirrer

 Thermometer

 TLC Plates

 Vials

3.1.2 Chemicals

 Acetic acid (Merck KGaA)

 Benzophenone (BDH)

 Chloroform (BDH)

 Citalopram Oxalate (Sigma-Aldrich)

 Dichloromethane (Sigma-Aldrich)
 34

 Distilled water (PCSIR)

 Ethanol (Merck-KGaA)

 Ethyl methyl ketone (Sigma-Aldrich)

 Iodine (BDH)

 Methanol (Merck KGaA)

 Saturated sodium carbonate (Merck KGaA)

 Sodium acetate (BDH)

 Sodium hydroxide (BDH)

 Sodium sulphate (Merck KGaA)

3.1.3 Instruments

 GC-MS

 MS-ESI

 FTIR spectrophotometer (Shimadzu)

 UV spectrophotometer (Shimadzu)

3.2 METHODOLOGY

3.2.1 Solution Preparation

0.1N solution of sodium hydroxide

 35

This was prepared by adding sodium hydroxide (0.4g) in water (100ml).

10% Sodium bi-carbonate solution

This was prepared by adding sodium bi-carbonate (10g) in water (25ml).

Saturated solution of sodium carbonate

This was prepared by adding sodium carbonate in water until no more sodium sulphate
was dissolved in water.

3.2.2 Solvent Extraction

Liquid–liquid extraction (LLE), also known as solvent extraction and partitioning, is a

method to separate compounds or metal complexes, based on their relative solubility in
two different immiscible liquids, usually water (polar) and an organic solvent (non-
polar).

Solvent Extraction by chloroform

Chloroform was used as an organic solvent for extraction of Escitalopram free base (1).

Solvent Extraction by Dichloromethane

Dichloromethane was used as an organic solvent for extraction of desmethylescitalopram

(4), didesmethylescitalopram (5) and its analogues (36 & 37).

3.2.3 Refluxing

Refluxing is a technique involving the condensation of vapors and the return of this
condensate to the system from which it originated. During preparation of intermediate
and analogues compound 36 and 37, refluxing was carried out.

3.2.4 Distillation

Distillation is a method of separating the constituents of mixture (either a liquid or

gaseous one). It is a physical process and not a chemical reaction using the different
 36

boiling temperatures of the constituents to separate them from the others. So, it was
carried out for concentrating all the resulting solutions.

3.2.5 Thin layer chromatography

It is based on the principle of adsorption chromatography or partition chromatography or

combination of both, depending on adsorbent, its treatment and nature of solvents
employed. The components with more affinity towards stationary phase travels slower
and components with less affinity towards stationary phase travel faster. TLC was
performed for confirmation of intermediate and its analogues. Solvent system used was
Chloroform: Methanol.

3.3 EXPERIMENTAL WORK

3.3.1 Purification of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-

1,3-dihydroisobenzofuran-5-carbonitrile oxalate (35)

(1)

Escitalopram oxalate (35, 5g, 12.06mmol) was dissolved in water (30 mL). For
converting it into free base (1), saturated solution of sodium carbonate was used to
maintain pH of solution to 8-9. The extraction of the compound was done with
chloroform. The organic layer was separated and kept in anhydrous sodium sulphate for
 37

overnight and filtered. The resulting solution was concentrated by distillation. Crude
material was confirmed by Thin Layer Chromatography (TLC). The solvent system used
was 1:9, v/v for methanol: chloroform respectively. UV chamber (short UV - 254nm)
was used to visualize spots. The escitalopram free base (1) was colorless thick oil,
weighed and stored at room temperature. The yield obtained was 4.84g (96.8%).

3.3.2 Synthesis of (S)-1-(4-fluorophenyl)-1-(3-methylamino)propyl)-1,3-

dihydroisobenzofuran-5-carbonitrile (4)

(4)

Escitalopram free base (1, 1.90g, 5.85mmol) was dissolved in ethanol (10mL). Then
sodium acetate (3.32g, 78.9mmol) was added to reaction mixture and stirred until
compound was dissolved completely. Further iodine (1.26g, 9.92mmol) was added at
regular intervals. pH of solution was checked and adjusted between 8.5-9.5 by adding
0.1N sodium hydroxide in drops. The reaction was continued, pH and Thin Layer
Chromatography (TLC) were checked every hour. The progress of reaction was checked
by Thin Layer Chromatography (TLC) using Methanol and Chloroform in ratio of
0.2:9.8,v/v as solvent system. The spots were visualized and observed by UV light at 254
nm. Reaction was continued for 48 hours until the clear solution was formed. After the
completion of reaction, 100mL of dichloromethane was used for solvent extraction. In
this way organic and aqueous layers were extracted. Organic layer was separated and
washed by using saturated solution of sodium sulphate, kept overnight in anhydrous
 38

sodium sulphate. Filtered it and was concentrated by distillation. The dark brown thick
oil was weighed. The yield obtained was 1.80g (94.7%).

ES-MS m/z: [M + H]+, 311

3.3.3 Synthesis of (S)-1-(3-aminopropyl)-1-(4-fluorophenyl)-1,3-

dihydroisobenzofuran-5-carbonitrile (5)

(5)

Desmethylescitalopram (4, 1.72g, 5.80mmol) dissolved in 10 mL of ethanol in a round

bottom flask (100 mL). Sodium acetate (3.32g, 78.9mmol) was mixed to the reaction
mixture and blended until compound dissolved completely. Iodine (1.26g, 9.92mmol)
was added at regular intervals into it. The pH of solution was adjusted between 8.5-9.5
by adding 0.1N sodium hydroxide drop wise. The reaction was continued and the pH and
TLC were checked every hour. Reaction was continued for 48 hours until the clear
solution was formed. The progress of reaction was checked by Thin Layer
Chromatography (TLC) using Methanol and Chloroform in ratio of 0.2:9.8,v/v as solvent
system. The spots were visualized and observed by UV light at 254 nm. After the
completion of reaction, 100mL of dichloromethane was used for solvent extraction. The
organic layer was separated and washed with saturated solution of sodium sulphate thrice
and kept in anhydrous sodium sulphate overnight. The resulting solution was filtered and
concentrated by distillation. The dark brown thick oil was obtained. The yield obtained
was 1.59 (92.44%).

ES-MS m/z: [M + H]+, 195

 39

3.3.4 Derivatives of (S)-1(3-aminopropyl)-1-(4-fluorophenyl)-1,3-

dihydroisobenzofuran-5-carbonitrile

N, N-didesmethylescitalopram‟s derivatives (5) were prepared with aliphatic and

aromatic ketones i.e; ethyl methyl ketone and bezophenone respectively.

3.3.4.1 Synthesis of (S,E)-1-(3-(butan-2-ylideneamino)propyl)-1-(4-fluorophenyl)-

1,3-dihydroisobenzofuran-5-carbonitrile (36)

(36)

Didesmethylescitalopram (5, 0.4g, 1.34mmol) was dissolved in ethanol (10mL). Initially

heating and stirring of solution was carried out for 5-10 minutes. Ethylmethyl ketone
(0.12mL) was added into it. Later, 3-5 drops of acetic acid were also added. Refluxing
continued for 2 hours at 78.380C temperature. The reaction was monitored by TLC. The
pH of the solution was maintained using 10% solution of sodium bi-carbonate. The
extraction of the compound was done with dichloromethane. The organic layer was dried
with anhydrous sodium sulphate and then filtered. The resulting solution was kept on
slow evaporation at room temperature. The dark brown thick oil was obtained. The yield
obtained was 0.35g (87.5%).

UV (λmax) nm 235

FTIR (cm-1) 1683.86, 1083.99, 1338.60, 2270.50, 1373.32, 1643

 40

3.3.4.2 Synthesis of (S)-1-(3-((diphenylmethylene)amino)propyl)-1-(4-fluorophenyl)-

1,3-dihydroisobenzofuran-5-carbonitrile (37)

(37)

Didesmethylescitalopram (5, 0.4g, 1.34mmol) was dissolved in ethanol (10mL). Initially

heating and stirring of solution was carried out for 5-10 minutes. Then benzophenone
(0.24g, 1.31mmol) was added into it. Subsequently, 3-5 drops of acetic acid were also
added. The refluxing continued for 3 hours at 78.380C temperature. The reaction mixture
was analyzed by TLC. The pH of the solution was maintained using 10% solution of
sodium bi-carbonate. The extraction of the compound was done with dichloromethane.
The organic layer was separated and dried by using anhydrous sodium sulphate and then
filtered. The resulting solution was then allowed to slow evaporate at room temperature.
The dark brown thick oil was obtained. The yield obtained was 0.37g (92.5%).

UV (λmax) nm 245

FTIR (cm-1) 1683.86, 1087.85, 1280.73, 2222, 1087.85, 1506.41

3.4 ACETYLCHOLINESTERASE ASSAY

The AChE inhibition activity was accomplished by the reported method with slight
alterations. The reaction mixture consisted of 100 µL of the total volume. It contained
Na2HPO4 buffer 60 µL with pH 7.7 and 50 mM concentration. The test compound (0.5
mM well-1) ten µL was incorporated, then 10 µL (0.005 unit well-1) enzyme was added.
The reaction contents were thoroughly mixed and at 405 nm pre-read. Then at 37 ºC for
 41

ten minutes the contents were pre-incubated. The reaction started when 0.5 mM well-1
substrate (acetylthiocholine iodide) 10 µL was added, and DTNB (0.5 mM well -1) of 10
µL. The absorbance was recorded at 405 nm by 96-well plate reader Synergy HT, Biotek,
USA at 37ºC after half an hour of incubation. All experiments were performed with their
respective standards (controls) in triplicate. As a positive control Eserine (0.5 mM well -1)
was practiced. The percent inhibition was measured by following equation.

Inhibition (%) = (Control – Test ⁄ Control) x 100

The IC50 values were analyzed by EZ–Fit Enzyme kinetics software (Perrella Scientific
Inc. Amherst, USA).

3.5 BUTYRYLCHOLINESTERASE ASSAY

The BChE inhibition activities have been achieved by already described method with
small modifications and BChE (Sigma Inc.) enzyme was used in this method.
 42

CHAPTER-4

RESULTS AND DISCUSSION

Figure: 4.1 Schematic overview of the research work.

 43

4.1 THIN LAYER CHROMATOGRAPHY

Figure: 4.1 TLC of Compound 5

 44

Figure: 4.2 TLC of compound 36

 45

Figure: 4.3 TLC of compound 37

 46

Table: 1

Physical data of compounds

Compound Structural Color Solubility Molecular Yield

number formula weight
%
(g/mol)

2 C20H21FN2O Light Ethanol/Methanol/ 324.17 96.8

yellow
chloroform

5 C18H17FN2O Dark Ethanol/Methanol/ 296.34 92.44

brown
dichloromethane

36 C22H23FN2O Brown Ethanol/Methanol/ 350.18 87.5

dichloromethane

37 C31H25FN2O Brown Ethanol/Methanol/ 460.20 92.5

dichloromethane
 47

4.1 MASS SPECTRA OF DESMETHYLESCITALOPRAM (4)

The molecular formula of desmethylescitalopram (4) is C19H19FN2O and it shows peak at

311.

[M+H]+=311

[310+ 1]+= 311

Figure: 4.4 MS ESI of desmethylescitalopram (4)

 48

4.2 MASS SPECTRA OF DIDESMETHYLESCITALOPRAM (5)

The molecular formula of Didesmethylescitalopram (5) is C18H17FN2O and it shows peak

at 195.

[M + H]+= 195

Figure: 4.5 MS ESI of Didesmethylescitalopram (5)

 49

4.3 ULTRAVIOLET-VISIBLE SPECTRUM OF COMPOUND 36

In UV spectra absorbance and wavelength are taken at vertical and horizontal axis
respectively. Compound 36 show maximum absorption at relatively sharp peak at
235nm. It is measured conveniently due to the region of spectrum.

Figure: 4.6 UV spectra of compound (36) λmax =235nm

 50

4.4 ULTRAVIOLET-VISIBLE SPECTRUM OF COMPOUND 37

In UV spectra absorbance and wavelength are taken at vertical and horizontal axis
respectively. Compound 37 showed maximum absorption at relatively sharp peak at
245nm. It is measured conveniently due to the region of spectrum.

Figure: 4.7 UV spectra of compound (37) λmax=245nm

 51

4.5 FTIR ANALYSES OF COMPOUND 36

The synthesized compound 36 was characterized by FTIR which determined the bonding
of introduced benzyledene group to intermediate. Peak at 1338.60cm-1 indicated presence
of C─N. C─C stretch exists at peak of 1083.99cm-1. Peak at 1683.86cm-1 is attributable to
stretching of N═C bond present in shiff‟s base. A peak at 1373.32cm-1 is assigned to
C─F stretching and at 1643cm1 peak for C═C is observed. Peak at 2270 indicated
presence of C≡N. No peak for N−H at 3400cm-1 was observed in spectra which
confirmed the replacement of N−H bond by N═C group. The FTIR spectrum confirmed
the formation of compound 36.

Figure 4.8 FTIR Spectra of Compound 36

 52

Table: 2

FTIR spectrum data of compound 36

Serial Number Functional Frequency

groups
(cm-1)

Spectrum Value Literature Value

1 N═C 1683.86 1640-1690

2 C−C 1083.99 1130

3 C−N 1338.60 1342-1266

4 C≡N 2270.50 2225

5 C−F 1373.32 1400-1000

6 C═C 1643 1648-1638

7 N−H - 3400
 53

4.6 FTIR ANALYSES OF COMPOUND 37

The synthesized compound 37 was characterized by FTIR which determined the bonding
of introduced benzyledene group to intermediate. Peak at 1280.73cm-1 indicated presence
of C─N. A C─C stretch exists at peak of 1087.85cm-1. Peak at 1683.86cm-1 is attributable
to stretching of N═C bond present in shiff‟s base. A peak at 1087.85cm-1 is assigned to
C─F stretching and at 1506.41cm1 peak for C═C is observed. Peak at 2222 cm-1 indicated
presence of C≡N. No peak for N−H at 3400cm-1 was observed in spectra which
confirmed the replacement of N−H bond by N═C group. The FTIR spectrum confirmed
the formation of compound 37.

Figure 4.9 FTIR Spectra of Compound 37

 54

Table: 3

FTIR spectrum data of compound 37

Serial Number Functional Frequency

groups
(cm-1)

Spectrum Value Literature Value

1 N═C 1683.86 1640-1690

2 C−C 1087.85 1130

3 C−N 1280.73 1342-1266

4 C≡N 2222 2225

5 C−F 1087.85 1400-1000

6 C═C 1506.41 1480-1600

7 N−H - 3400
 55

4.7 BIOLOGICAL EVALUATION

The biological screening of a novel series of synthesized didesmethylescitalopram (5)

and its derivatives (36-37) for cholinesterase inhibition was executed by Ellman method
using Eserine as positive control. The inhibitor interacts with AChE/BChE, and avoids
the hydrolysis of acetyl/butyrylcholine. The inhibitor decreases the enzyme activity
which is associated to the inhibitor concentration. The unbound enzyme hydrolyzes the
acetyl/butyrylthiocholine (ASCh/BSCh) and finally its quantity is calculated, by
quantification of the product of its reaction with DTNB (5, 5′-dithiobis-(2-nitrobenzoic
acid)) spectrophotometrically.

4.7.1 Acetylcholinesterase Inhibition

In this study, a series of synthesized didesmethylescitalopram (5) and its derivatives (36-
37) have been screened for AChE inhibition activities (Table 4). The results of this study
indicate that ligand 36 is the most potent compound of the series having IC50 = 1.09 μM
which is 27-fold less active than the standard eserine (IC50 = 0.04±0.0001). Other
member of the series has shown good inhibition at 3.71 μM. The intermediate ligand
desmethylescitalopram (4) also exhibited good AChE inhibition activity at IC50 = 6.85
μM.

Table: 4

IC50 values of AChE for didesmethylescitalopram (5) and its derivatives (36-37).

Serial Compound Concentration Acetylcholinesterase

No. No. (mM) % Inhibition IC50 (µM)

1 5 0.5 89.15±0.19 6.85±0.33

2 36 0.5 95.51±0.51 1.09±0.11
3 37 0.5 90.07±0.15 3.71±0.32
Eserine 0.5 91.27±1.17 0.04±0.0001
Standard
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Results are the mean of three independent experiments (n = 3) ± S.D.

4.7.2 Butyrylcholinesterase Inhibition

BChE inhibition of a novel series of a series of synthesized didesmethylescitalopram (5)

and its derivatives (36-37) have been determined and their IC50 values are calculated
(Table 5). The ligand 36 has been found to be the most potent compound of the series
with lowest IC50 = 1.11 μM. While ligand 37 of the series has depicted IC50 value 2.90
μM. The intermediate compound didesmethylescitalopram (5) also showed good BChE
activity at 4.08 μM. All these compounds exhibiting selectivity from 0.98- to < 1.68-fold
more towards BChE than AChE.

Table: 5

IC50 values of BChE for didesmethylescitalopram (5) and its derivatives (36-37).

Serial Compound Concentration Butyrylcholinesterase Selectivity for

Number Number (mM) % IC50 (µM)a Butyrylcholinesterase
Inhibition
1 5 0.5 87.52±0.63 4.08±0.27 1.68
2 36 0.5 90.09±0.34 1.11±0.31 0.98
3 37 0.5 88.01±0.41 2.90±0.66 1.28
Eserine 0.5 82.82±1.09 0.85±0.0001
Standard

Results are the mean of three independent experiments (n = 3) ± S.D., Selectivity for
BChE = IC50 (ACHE)/ IC50 (BChE).
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CONCLUSION

In present research work, Didesmethylescitalopram (5) was synthesized by refluxing

escitalopram with iodine and its analogues were prepared by refluxing this compound (5)
with aliphatic and aromatic ketones. The synthesized compounds were obtained as thick
brown oils with better yield and were characterized by GC-MS, MS-ESI, FTIR and UV
spectroscopy. The cholinesterase activity was evaluated for didesmethylescitalopram (5)
and its analogues (36 & 37). Didesmethylescitalopram (5) showed percentage inhibition
of 89.15 ±0.19 and 87.52 ±0.63 with IC50 values of 6.85 ±0.33μM and 4.08 ±0.27μM for
acetylcholinesterase and butyrylcholinesterase respectively. The compound 36 depicted
95.51±0.51% (IC50, 1.09μM) AChE inhibition while 90.09±0.34% (IC50, 1.11±0.31μM)
BChE inhibition. Likewise, compound 37 showed 90.07±0.15% (IC50, 3.71±0.32μM)
AChE inhibition while 88.01±0.41 (IC50, 2.90±0.66μM) BChE inhibition. So, compound
36 is the most potent compound of the series possessing highest cholinesterase inhibition.