A novel potential therapeutic avenue for autism: Design, synthesis and pharmacophore generation of SSRIs with dual action

Article abstract of DOI:10.1016/j.bmcl.2011.09.046

Autism symptoms are currently modulated by Selective Serotonin Reuptake Inhibitors (SSRIs). SSRIs slow onset of action limits their efficiency. The established synergistic activity of SSRIs and 5HT_1B/1D autoreceptors antagonists motivated us to incorporate SSRIs and 5HT_1B/1D antagonists in one 'hybrid' molecule. A library of virtual 'hybrid' molecules was designed using the tethering technique. A pharmacophore model was generated derived from 16 structurally diverse SSRIs (K_i = 0.013-5000 nM) and used as 3D query. Compounds with fit values (≥2) were chosen for synthesis and subsequent in vitro biological evaluation. Our pharmacophore model is a promising milestone to a class of SSRIs with dual action.

Full text of DOI:10.1016/j.bmcl.2011.09.046

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6714–6723
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel potential therapeutic avenue for autism: Design, synthesis
and pharmacophore generation of SSRIs with dual action
Ola M. Ghoneim a, , Diaa A. Ibrahim b, Ibrahim M. El-Deeb c, So Ha Lee c, Raymond G. Booth d
⇑
a College of Pharmacy, Qatar University, PO Box 2713, Doha, Qatar
b National Organization for Drug Control and Research, PO Box 29, Cairo, Giza 112311, Egypt
c Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
d College of Pharmacy, University of Florida-Gainesville, PO Box 10048, 1600 SW Archer Road, Gainesville, FL 32610, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2011
Revised 3 September 2011
Accepted 15 September 2011
Available online 21 September 2011
Autism symptoms are currently modulated by Selective Serotonin Reuptake Inhibitors (SSRIs). SSRIs slow
onset of action limits their efficiency. The established synergistic activity of SSRIs and 5HT1B/1D autore-
ceptors antagonists motivated us to incorporate SSRIs and 5HT1B/1D antagonists in one ‘hybrid’ molecule.
A library of virtual ‘hybrid’ molecules was designed using the tethering technique. A pharmacophore
model was generated derived from 16 structurally diverse SSRIs (Ki = 0.013–5000 nM) and used as 3D
query. Compounds with fit values (P2) were chosen for synthesis and subsequent in vitro biological eval-
uation. Our pharmacophore model is a promising milestone to a class of SSRIs with dual action.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Autism
Selective Serotonin Reuptake Inhibitors
(SSRIs)
Pharmacophore
Autism is a poorly understood neurodevelopmental disorder
that was first described by Kanner in 1943.1 Autism prevalence
has significantly increased worldwide from 1 case per 10,000 chil-
dren in the early 1990’s to reach 1 case per 110 children in 2010
according to the Centers for Disease Control and Prevention.2
Autism manifests itself by three main domains; social skills defi-
cit, communication impairments, and repetitive behaviors. Selec-
tive Serotonin Reuptake Inhibitors (SSRIs) are currently the drug
class of choice for treating autism symptoms including repetitive
behaviors.3 SSRIs are originally synthetic antidepressant agents
with established efficacy for the management of anxiety, obses-
sive compulsive disorder (OCD) and recently employed for reduc-
ing or modulating repetitive behavior symptoms in autistic
children.4 As a major drawback, it takes 3–6 weeks for SSRIs to be-
come therapeutically efficient. The delay in efficiency varies from
one individual to another and significantly decreases compliance,
and increases anxiety level about the effectiveness of the under-
going therapy.5 An explanation for the delay in efficiency is attrib-
uted to the mechanism by which SSRIs operate. SSRIs permeate
across the blood–brain barrier and inhibit the neurotransmitter
serotonin (5-HT) binding to its transporter, which blocks 5-HT
reuptake and increase its concentration in the synaptic cleft
(Fig. 1, Panel A). The elevated concentration of 5-HT in the
synaptic cleft stimulates postsynaptic 5-HT receptors to initiate
a cascade of pharmacological effects that relieve autism symp-
toms including repetitive behaviors and restricted interests.6
However, the elevated concentrations of 5-HT in the synaptic cleft
also stimulates inhibitory presynaptic 5-HT autoreceptors (e.g.,
5-HT1B/1D). Inhibitory receptors, by definition, regulate the release
of 5-HT in an inhibitory manner and negatively affect the mode of
action of SSRIs and delay the therapeutic effect of SSRIs for several
weeks after their administration.4,5 Acute blockade of the inhibi-
tory autoreceptors proved to eliminate their negative feedback ef-
fect on 5-HT release and synergistically increased the efficiency of
SSRIs (Fig. 1, Panel B). Accordingly, co-administration of SSRIs
with 5-HT1B/1D receptor antagonists is advantageous over SSRIs
alone with respect to the magnitude of extracellular brain 5-HT
levels produced.6,7 The established synergistic activity of SSRIs
and autoreceptors antagonists motivated us to incorporate 5-HT
reuptake inhibitors and antagonists of the 5-HT1B/1D autoreceptors
in one molecule to develop ‘hybrid’ SSRIs with dual action as anti-
autism candidates. We hypothesized that ‘‘hybrid’’ anti-autism
drug candidates will exhibit fast and efficient inhibition of 5-HT
reuptake, which will result in a rapid and consistent increase in
5-HT concentration in the synaptic cleft and a corresponding
quick control over autism symptoms. Development of hybrid
anti-autism drugs will eliminate the need for co-administration
of SSRIs and 5-HT autoreceptor antagonists and the associated
high risk of dual pharmacokinetics/dosage/side effect and poor
⇑
Corresponding author at present address: Saint Joseph College, School of
Pharmacy, 229 Trumbull street, Hartford, CT 06103, USA. Tel.: +860 231 6894; fax: +
860 231 5759.
E-mail address: oghoneim@sjc.edu (O.M. Ghoneim).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.046

O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
6715
Figure 1. The mechanism of action of SSRIs in the presence/absence of autoreceptor antagonists. The green arrows show the two pathways regulating serotonin (5-HT)
concentration in the synaptic cleft through the reuptake transporter and autoreceptor. In panel A, administration of SSRIs alone allows the autoreceptors to inhibit 5-HT
release, which delays the therapeutic effect of SSRIs. In panel B, co-administration of SSRIs and autoreceptor antagonists abolish the inhibitory effect of the autoreceptors and
synergistically increase the therapeutic effect of SSRIs.
MeO
O
MeO
O
N
N
H
N
N
H
N
OCH3
N
N
N
N
N
O
GR-127935
non-selective 5-HT1B/1D antagonist
Lead compound (Huang et al., 2005)
selective 5-HT1B antagonist
N
O
N
N
O
Cl
O
N
N
N
O-tolyl-piperazide
selective 5-HT1B antagonist
BRL-15,572
selective 5-HT1D antagonist
Figure 2. Chemical structures of known 5-HT1B/1D antagonists.

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O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
as an antagonist for 5-HT1D receptor (Fig. 2).11 By critically analyz-
ing the structure of these 5-HT1B/1D antagonists, one can recognize
that the N-(4-methoxyphenyl)amide group with and without the
4-methylpiperazinyl moiety (red group, Fig. 2) to be a common
molecular scaffolds in GR127935, Huang’s lead compound, and
others (not shown). Substitution of the carboxylic side of the amide
group tolerates a wide variety of bulky, aromatic, and nonaromatic
substituents without affecting the binding affinity of these mole-
cules to 5HT1B/1D receptors.12 Earlier studies clearly showed that
substitution with functionalized arylpiperazine groups into a series
of nonselective 5-HT1 ligands resulted in their selective binding to
5-HT1B/1D receptors versus other 5-HT1 subtypes.12 These results
together with earlier findings suggest that 5-HT1B/1D receptors pos-
sess a deep binding pocket in the binding domain that recognizes
the bulky substitution with the arylpiperazine group on the seroto-
nin ring. Specifically, this region of bulk tolerance seems to differ-
entiate between different 5-HT1 receptor subtypes and allows
preferential binding to 5-HT1B/1D receptor subtypes (Fig. 2).6,12
From the above, we can conclude that both N-(4-methoxy-
phenyl)amide group with/without 4-methylpiperazine and func-
tionalized arylpiperazine moieties are promising molecular
scaffolds for development of 5-HT1B/1D selective antagonists (red
group in Figs. 2 and 4). We have utilized these moieties to function
as selective antagonists for 5-HT1B/1D receptors in our ‘hybrid’
molecules.
Figure 3. Chemical structures of known 5-HT reuptake inhibitors.
overall patient compliance. In our journey to discover hybrid anti-
autism candidates, we report herein the design, pharmacophore
generation and the chemical synthesis of ‘hybrid’ SSRIs with dual
action. To the best of our knowledge, this is an unprecedented
therapeutic avenue for modulating autism symptoms.
Our approach for developing ‘hybrid’ small molecules with dual
action is based on the tethering technique (Fragment based drug
discovery).8 Ideally, we identify the molecular scaffold(s) responsi-
ble for the desired pharmacological action(s) and link the two
scaffolds together via an appropriate covalent linker.
Molecular scaffolds of 5-HT reuptake inhibitors
Compared to 5-HT1B/1D antagonists, examining the chemical
structure of SSRIs did not reveal an obvious structural feature that
may be responsible for their activity. The wide structural diversity
of SSRIs (Fig. 3) suggests that their binding interaction to the Sero-
tonin Reuptake Transporter (SERT) is quite flexible.13 By analyzing
the structure of sertraline and paroxetine (Fig. 3), one easily recog-
nizes that sertraline has a halogenated phenyltetralin moiety with
2 chiral centers, whereas the main nucleus of paroxetine is a halo-
genated phenylpiperidine with one chiral center. One approach to
obtain a more rigid conformational analog and eliminate the chiral
centers is isosteric replacement of the phenyltetralin ring of sertr-
alin and phenylpiperidine ring of paroxetine by phenylquinoxaline
and phenylpiperazine, respectively, while maintaining the halogen
substitution, which is essential for maintaining SSRI pharmacolog-
ical activity.14 Other reports showed that positions 6 and 7 of
Molecular scaffolds of 5-HT1B/1D Antagonists
GR127935 was the first reported potent 5-HT1B/1D antagonist
(Fig. 2), followed by several other potent compounds.9 Few ligands
were successfully synthesized with selectivity towards either the
5-HT1B or 5-HT1D receptor subtype. For example, O-tolylpiperazide
and the lead compound reported by Huang et al. are specific antag-
onists for the 5-HT1B receptor (Fig. 2).10 BRL-15,572 was developed
MeO
O
O
O
N
N
N
N
H
N
N
CF3
N
Cl
Series A
H
Series B
Cl
O
N
N
O
N
N
O
O
CF3
MeO
N
N
N
Cl
Series C
Series D
Cl
Figure 4. Representative examples of virtual hybrid SSRI with dual action.

O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
6717
NH
O
N
O
N
Cl
Cl
HN
O
N
O
O
1
NH2
N
H
N
3
2
HN
O
S
S
4
O
O
HN
O
N
S
N
N
O
N
N
F
6
N
O
5
H2N
N
7
NH2
O
HO
N
N
Cl
N
S
N
O
S
O
N
N
F
9
I
NH2
10
F
OH
NH2
8
11
N
O
N
O
S
NH
H
N
N
N
N
HO
N
NH2
12
NH
O
N
F
O
13
O
O
14
H
N
O
F
15
F
F
F
F
16
Figure 5. Literature based SSRIs used as training set in building the pharmacophore model.
sertraline and the equivalent position 3 of paroxetine can tolerate
bulkier polar electron withdrawing groups such as carboxamide
while maintaining their SSRI pharmacological activity.14 On the
other hand, the main nucleus of Fluoxetine, which is another
known SSRI, is benzyloxy halogenated benzene (Fig. 3).13 Similarly,
the main nucleus of citalopram is benzyloxymethyl halogenated
benzene (Fig. 3). Results show that the side chain of aliphatic
amine might not be a requirement for SSRI activity since amine
chains with different length (ethane, propane, butane) have shown
SSRI activity but the basic nitrogen is a requirement for SSRI activ-
ity.13 Based on these studies, we believe that both pheynlquinoxa-
conferring SSRI pharmacological activity in our ‘hybrid’ molecules
(blue groups in Figs. 3 and 4).
Design virtual hybrid SSRIs with dual action
Tethering technique simply link two molecular scaffolds be-
lieved to be responsible of the desired pharmacological effect
through an appropriate covalent linker with the assumption that
the linker is easily metabolized inside the body into its two compo-
nents. Accordingly, mix and match the four previously described
molecular scaffolds (red and blue groups, Figs. 2 and 3) will result
in minimum of four different series of virtual hybrid compounds
(series A–D; Fig. 4).
line ring with carboxamide in position
7 and benzyloxy
halogenated benzene represent promising molecular scaffolds for
Figure 6. Pharmacophore model used in the selection of the virtual hybrid compounds includes two hydrophobic centers (cyan color) and one hydrogen bond donor (HBD;
purple color).

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O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
O
N
N
O
N
F
F
O
1
F
N
O
2
O
N
N
N
H
N
N
N
O
F
F
O
O
F
4
3
O
S
N
H
N
N
F
N
O
F
F
F
F
O
O
F
6
S
5
N
Cl
N
N
N
O
O
OH
8
7
O
F
N
N
O
9
Figure 7. Inactive SSRI compounds used to add exclusion spheres to the pharmacophore model.
The selection of promising compounds for chemical synthesis
being tested using the same binding assay. Wide range of binding
affinities (Ki) was chosen, ranging from 0.013 to 5000 nM. Struc-
tural information from the training set identified a set of features
crucial for activity and was considered to represent a pharmaco-
and subsequent in vitro biological evaluation from the generated
virtual hybrid library required a computerized cutoff value. This
led us to generate pharmacophore models using the existing SSRIs
from the literature as well as our proposed SSRIs with dual action
to gain further insights and help determine the selection of the
compounds to the nest phase.
Table 1
Some proposed compounds and their Fit Values
Common pharmacophore features
Compound ID
Compound
Fit Value
Sixteen compounds (Fig. 5) were collected from the literature
and used as a training set in the pharmacophore building. Criteria
for compound selection include diverse molecular structure and
RHO-001
Not mapped
RHO-002
RHO-003
Not mapped
2.100
RHO-004
RHO-005
2.304
2.208
Figure 8. Sterically-refined versions of our pharmacophore with 68 added exclu-
sion volumes model.

O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
6719
color) associated to its protein acceptor site and acceptor atom
(Fig. 6). The interfeature distances were considered to be 9.93,
10.59 and 4.03 Å for distances between the hydrophobic center 2
and the hydrogen bond donor, the hydrophobic center 2 and the
hydrophobic center 1, the hydrophobic center 1 and hydrogen
bond donor, respectively (Fig. 6).
Only one angle constraint was used for the hydrophobic and the
donor atom features, thus allowing the hydrophobic centers to
cover a larger domain. Since not all proposed hybrid compounds
place hydrophobes in both regions, a partial match directive was
used on the query for the hydrophobic centers to match com-
pounds that contain only one.
Table 1 (continued)
Compound ID
Compound
Fit Value
2.212
RHO-010
Addition of exclusion volumes
RHO-011
Not mapped
Although ligand-based pharmacophores serve as excellent tools
to probe ligand/macromolecule recognition and can serve as useful
3D-QSAR models and 3D search queries, they suffer from a major
drawback: They lack steric constrains necessary to define the size
of the binding pocket. This liability renders pharmacophoric mod-
els rather promiscuous. Therefore, we decided to complement our
selected pharmacophore model with exclusion spheres. Excluded
volumes resemble sterically inaccessible regions within the bind-
ing site. HipHop-Refine requires a list of inactive training com-
pounds (Fig. 7) together with two qualitative descriptors that
characterize the way by which each training compound contrib-
utes in defining the exclusion space (Principal and MaxOmit-
Feat).18,19 All the nine inactive compounds, which used in adding
the steric volumes together with their HipHop-Refine parameters,
have 0 as their principal value and 2 as their maximum omitted
features. Figure 8 shows the final pharmacophore with 68 added
exclusion volumes. Using this generated pharmacophore model,
we were able to map our proposed hybrid anti-autism compounds
into the model to locate the subset of promising compounds that
are capable of binding to SERT with a similar set of interactions. Fi-
nally, the proposed compounds with fit values (P2) were selected
for chemical synthesis and biological evaluation (Table 1). Figure 9,
10 and 11 demonstrate the mapping of compounds RHO-003, RHO-
004 and RHO-0012 to the generated pharmacophore with fit values
2.10, 2.30 and 1.98 respectively.
RHO-012
1.979
phore hypothesis. Based on previous experience15
, HypoGen
module in Discovery Studios DS 2.016,17 was used to generate our
pharmacophore models wherein it evaluates a collection of confor-
mational models for all compounds, and maps them to the selected
crucial features.
The top ranked pharmacophore model is expected to identify
the common binding features and the hypothetical orientation of
the active compounds interacting with their target. Our model is
represented by two hydrophobic centers (Hydrophobic 1, Hydro-
phobic 2; cyan color) and one hydrogen bond donor (HBD; purple
RHO-003
Fit Value: 2.10
F
F
F
O
O
O
N
H
Figure 9. Mapping compound RHO-003 to the sterically-refined versions of our pharmacophore model (Fit Value = 2.10).

6720
O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
RHO-004
Fit Value: 2.30
N
F
O
F
N
O
F
H
Figure 10. Mapping compound RHO-004 to the sterically-refined versions of our pharmacophore model (Fit Value= 2.30).
ent primary amines in the presence of EDC/HoBt to afford the
Results
target compounds RHO-003 to RHO-005 in good yields (71–82%).
In Scheme 2, 4-fluoro-3-nitrobenzoic acid was converted to the
corresponding ester RHO-006, which was then subjected to Pd-cat-
alyzed coupling reaction with 3,4-dichloroaniline to give RHO-007.
Reduction of the nitro group following by reaction with 2-chloro-
acetyl chloride yielded the open ring analog of RHO-009, which
was then cyclized to the corresponding quinoxalinone ring RHO-
010. Methylation of the cyclic amide gave RHO-011, which was
then hydrolysed to the free carboxylic acid, converted to its acid
Proposed compounds that met the pharmacophore criteria (Fit
Value P2) were synthesized as candidates for pharmacological
assessment. Scheme 1 and Scheme 2 represent the successful syn-
thetic pathways to synthesize compounds RHO-001-RHO-012. In
Scheme 1, RHO-001 was obtained by coupling 4-trifluorophenol
with 2-bromo2-phenylacetate in the presence of an inorganic base.
Hydrolysis of the methyl ester of RHO-001 yielded the free carbox-
ylic acid RHO-002, which was then subjected to couple with differ-
Figure 11. Mapping compound RHO-012 to the sterically-refined versions of our pharmacophore model (Fit Value = 1.98).

O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
6721
O
O
Br
OH
O
O
O
(a)
F3C
+
CF3
RHO-001
(b)
O
O
O
NH2
O
N
OH
H
N
F3C
N
F3C
(c)
RHO-002
RHO-005
NH2
OCH3
OCH3
(c)
N
H2N
(c)
O
O
N
H
N
F3C
O
O
N
H
RHO-004
F3C
RHO-003
Scheme 1. Reagents and conditions: (a) K2CO3, DCM, Reflux, 6 h; (b) NaOH, MeOH, rt, 2 h; (c) EDCI, HoBT, DCM, rt, 24 h.
O
NH2
NO2
NH
O
O
O
Cl
NO2
F
NO2
F
Cl
HO
O
(a)
(b)
(c)
Cl
RHO-006
RHO-007
O
Cl
O
O
H
N
H
N
O
NH2
O
O
O
O
O
Cl
N
NH
NH Cl
Cl
(f)
(e)
(d)
Cl
Cl
Cl
Cl
Cl
RHO-008
O
RHO-009
RHO-010
O
Cl
O
N
N
N
O
N
N
O
O
(g, h, i)
N
H
N
O
N
NH2
Cl
Cl
N
RHO-011
RHO-012
Cl
Cl
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, rt, 24 h; (b) Cl2(PPh3)2Pd(II), Xantphos, K2CO3, Toluene, N2, reflux,12 h; (c) Pd/C, H2,THF, rt, 4 h; (d) TEA,
Dichloroethane, rt, 3 h; (e) KI, DBU, butanone, reflux, 4 h; (f) CH3I, NaH, DMA, N2, rt, 24 h; (g) NaOH, MeOH/H2O (4:1), reflux, 2 h; (h) SOCl2, DCM, reflux, 24 h; (i) 4-methoxy-
3-(4-methylpiperazin-1-yl)aniline, pyridine, DMAP, 60°, 20 h.

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O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
in vitro biological evaluation. Preliminary in vitro evaluation data
is promising and consistent with our prediction. All compounds
are undergoing further testing now and the full biological data will
be published in a future communication.
Acknowledgment
The authors would like to thank Dr. Brian Roth, Psychoactive
Drug Screening Program at University of North Carolina-Chapel
Hill23 for conducting the preliminary in vitro pharmacological
(affinity) assays. This publication was made possible by a grant
from the Qatar National Research Fund under its National Priorities
Research Program. Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of the
Qatar National Research Fund.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.046.
References and notes
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Figure 12. Representative concentration-response curves for RHO-012 displace-
ment of radioligand from human 5-HT1B (panel A) and 5-HT1D (panel B) receptors.
Displacement by the reference ligand ergotamine is shown for comparison.
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chloride analog and finally couple with 4-methoxy-3(4-methylpi-
perazin-1-yl)aniline to afford our final target compound RHO-012
in 56% yield.20
Although stuctural requirements for ligand binding at SERT are
highly variable, a basic amine moiety usually is present among
ligands with high-affinity for aminergic GPCRs—it is proposed that
the protonated amine can interact with the fully-conserved
aspartate residue at GPCR position 3.32.21,22 Accordingly, analog
RHO-012 was chosen to undergo preliminary pharmacological
assessment for affinity at human recombinant 5-HT1B and 5-HT1D
GPCRs expressed in HEK cell membranes.23 The radioreceptor com-
petition displacement assay used [3H]-CT (carboxytryptamine) and
[3H]-GR125743 to label 5-HT1B and 5-HT1D receptors, respectively.
Figure 12 shows competition displacement curves for RHO-012 in
comparison to the reference ligand ergotamine at 5-HT1B and
5-HT1D receptors; the Ki value for RHO-012 was 44 7.2 and
120 14 nM at 5-HT1B and 5-HT1D receptors, respectively. SERT
binding affinity is in progress.
13. Dorsey, J. M.; Miranda, M. G.; Cozzi, N. V.; Pinney, K. G. Bioorg. Med. Chem. 2004,
12, 1483.
14. Middleton, D. S.; Andrews, M.; Glossop, P.; Gymer, G.; Jessiman, A.; Johnson, P.
S.; MacKenny, M.; Pitcher, M. J.; Rooker, T.; Stobie, A.; Tang, K.; Morgan, P.
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19. Van Drie, J. H. Curr. Pharm. Des 2003, 9, 1649.
20. Synthesis of RHO-012 A mixture of RHO-011 (200 mg, 0.55 mmol) and NaOH
(440 mg, 11.0 mmol) in a mixed solvent of CH3OH and H2O (20 mL, 4:1 v/v)
was heated under reflux for 1 h. The reaction mixture was then filtered to
remove any insoluble impurities, and the resulted filtrate was concentrated in
vacuo, diluted with water (20 mL) and then neutralized with 50% HCl to pH 6.
The resulted precipitate was filtered, washed with cold water and left to dry in
air. The dried solids were dissolved in dichloromethane (20 mL), and to the
resulted solution was added thionyl chloride (0.08 mL, 1.1 mmol) in
a
dropwise manner. The resulted mixture was heated under reflux for 24 h,
after which, the solvent and excess thionyl chloride were removed under
vacuum. The residue was taken into THF (4 mL), and to this new solution was
added 4-methoxy-3-(4-methylpiperazin-1-yl)aniline (146 mg, 0.66 mg),
pyridine (1 mL) and a catalytic amount of DMAP, and the resulted mixture
was heated at 60 °C for 20 h. The reaction solvent was then removed under
vacuum, and the residue was purified by column chromatography (silica gel,
ethyl acetate/acetone 8:1 v/v) to give the pure product RHO-012 (190 mg,
56%),mp 123–124 °C; 1H NMR (CD3OD): d 2.35 (s, 3H, CH3), 2.63 (br s, 4H,
2CH2), 3.07 (br s, 4H, 2CH2), 3.42 (s, 3H, CH3), 3.83 (s, 3H, CH3), 4.24 (s, 2H,
CH2), 6.85–6.93 (m, 2H, ArH), 7.14 (dd, J = 2.3, 8.7 Hz, 1H, ArH), 7.29–7.37 (m,
3H, ArH), 7.45 (d, J = 8.6 Hz, 1H, ArH), 7.53 (d, J = 8.5 Hz, ArH), 7.69 (s, 1H, ArH),
7.90 (s, 1H, ArH); 13C NMR (CD3OD): 28.24, 44.71, 49.84, 51.80, 54.68, 54.90,
Conclusion
A library of virtual hybrid SSRI with dual action was designed.
Pharmacophore model was generated using structurally diverse
existing SSRIs with Ki range from 0.013–5000 nM. Exclusion vol-
umes were added to the chosen model to sterically refine it. The
sterically-refined version of the pharmacophore was generated
and used as 3D query for compound selection. Proposed com-
pounds with high fit values (P2) were selected for synthesis and

O. M. Ghoneim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6714–6723
6723
111.53, 112.19, 114.73, 115.18, 115.81, 121.91, 123.10, 123.67, 127.20, 127.61,
130.63, 130.96, 132.02, 132.81, 135.56, 136.94, 140.82, 143.33, 149.34, 165.84.
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