Serotonin transporter inhibitors: Synthesis and binding potency of 2'-methyl- and 3'-methyl-6-nitroquipazine

Article abstract of DOI:10.1016/S0960-894X(00)00546-1

Racemic 2'-methyl- and 3'-methyl-6-nitroquipazine ligands were selected as targets, synthesized and evaluated at the serotonin transporter employing an in vitro competitive inhibition assay with [³H]paroxetine and rat cortical membrane. The 2'-methyl-6-nitroquipazine was found to be 50 times more potent than the 3'-methyl-substituted counterpart and of comparable potency to the known high affinity agent 5-iodo-6-nitroquipazine. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00546-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2643±2646
Serotonin Transporter Inhibitors: Synthesis and Binding
Potency of 20-Methyl- and 30-Methyl-6-nitroquipazine
John M. Gerdes,a,* Steven C. DeFina,a Paul A. Wilsona and Scott E. Taylorb
aDepartment of Chemistry, Central Washington University, Ellensburg, WA 98926-7539, USA
bCenter for Functional Imaging, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA
Received 7 March 2000; accepted 15 September 2000
AbstractÐRacemic 20-methyl- and 30-methyl-6-nitroquipazine ligands were selected as targets, synthesized and evaluated at the
serotonin transporter employing an in vitro competitive inhibition assay with [3H]paroxetine and rat cortical membrane. The
20-methyl-6-nitroquipazine was found to be 50 times more potent than the 30-methyl-substituted counterpart and of comparable
potency to the known high anity agent 5-iodo-6-nitroquipazine. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
short-lived, radiolabels are placed often in¯uences the
radiotracer syntheses and stability and also receptor
anity and selectivity.11,12 Thus, many variables must
be considered in the development of a PET ligand.
Based on these criteria, the approach in our laboratory
towards developing new SERT PET agents has encom-
passed the use of the SERT pharmacophore models and
examination of select structure±anity relationship
(SAR) studies of potent SERT ligands.
The serotonin transporter (SERT) is an integral mem-
brane protein that is responsible for the reuptake
(transport) of the central nervous system biogenic amine
5-hydroxy-tryptamine (5-HT) from the synaptic cleft.1
Transport of 5-HT is sensitive to the presence of nano-
molar concentrations of SERT inhibitor ligands,2,3
including serotonin selective reuptake inhibitor drugs
(SSRIs). The SSRIs are thought to bind to the SERT4
at the same site as 5-HT or a closely overlapping site(s).5
The structural features of the SSRI agents are diverse,
encompassing agents with either one or two aromatic
structural components.2,6 These drugs along with other
SERT ligands have been utilized to develop a limited
number of SERT inhibitor pharmacophore models.7 9
The models describe regions in space for two distinct
aromatic moieties that are connected to a carbon back-
bone containing an amine atom terminus. In some
cases, one of the aromatic moieties may be joined to the
chain through a heteroatom linkage. The models7 may
be utilized as initial design constructs for the development
of new SERT inhibitors.
Of particular interest are SERT ligands that are com-
posed with only one aromatic structural component, a
heteroatom linkage and a minimum of other functional
groups. For example, analogue studies13 16 of the
established SERT inhibitor 6-nitroquipazine (6-nitro-2-
piperazinylquinoline, DU-24565),13,17 shown in Figure 1
as ligand 1, have provided the SERT selective 5-iodo-6-
nitroquipazine, ligand 2 (INQUIP). When evaluated in
vitro with rat cortical membrane SERT and competitive
inhibition binding conditions ([3H]paroxetine), agent 2
has been found to be a more potent ligand (inhibition
binding constant of 2, Ki=0.19Æ0.09 nM) than the
parent drug 1 (Ki=0.23Æ0.06 nM).16
Investigations to map the SERT in living brain have
employed positron emission tomography (PET) imaging
techniques.10,11 These studies have led to the discovery
of a limited number of PET imaging agents that are
appended with either ¯uorine-18 or carbon-11 positron-
emitting nuclides. The location on the ligands where the
Target Ligands
Our interest in the quipazine drug series has been to
identify locations on the parent drug 1 that would be
suitable for bearing an alkyl group ultimately contain-
ing a positron atom label. Since existing SERT phar-
macophore models of well known high anity SSRI
ligands describe regions for two distinct aromatic
*Corresponding author. Tel.: +1-509-963-2814; fax: +1-509-963-
1050; e-mail: gerdes@cwu.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00546-1

2644
J. M. Gerdes et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2643±2646
Syntheses
Preparation of the target ligands 3 and 4 followed the
synthetic routes shown in Scheme 1.20 22 The pathways
were patterned after related quipazine syntheses23,24 and
due to the preliminary nature of the investigation they
were e€ected in a racemic fashion. To produce ligand 3,
the more accessible 4-position of (rac)-2-methylpiper-
azine 5 (Lancaster Synthesis) was protected with tri-
phenylmethyl chloride25 to a€ord the amine 6. The
coupling of 6 with 2-chloroquinoline (Aldrich Chemical
Co.) at re¯ux temperature23 for an extended period
(125 h) in the presence of carbonate bu€er26 yielded the
adduct 7. Vigorous conditions were required for the
coupling, which were considered a consequence of the
steric demands imposed by the methyl group adjacent to
the 1-position amine reacting center of 6. Deprotec-
tion27 of intermediate 7 prꢀovided the amine 8. Regio-
speci®c nitration24 of 8 at 0 C a€orded the target ligand
3,28 in an overall 10% yield from starting material 5.
Formation of ligand 4 involved the coupling of 5 with
2-chloroquinoline to yield the amine 6. Nitration of 6
a€orded the target agent 4,29 in an overall yield of 55%.
Figure 1. Established serotonin transporter inhibitors (1 and 2) and
the target ligands (3 and 4).
moieties7,8 and inhibitor 6-nitroquipazine 1 contains
only one aromatic member (quinoline ring), then our
ligand design approach encompassed the placement of
an alkyl group at prospective locations where a second
aromatic moiety could be accommodated according to
the models. The 20- or 30-positions of the piperazine ring
of 1 were thought to be these structural locations.
Therefore, we sought to initially probe the SAR of
racemic 20- and 30-substituted variants of 6-nitroquipa-
zine, 1. The 20-methyl- and 30-methyl-6-nitroquipazines,
3 and 4, respectively, were chosen as target ligands.
Finally, analogues of 1 with substituents placed at these
positions are unknown.18
Binding Assessments
The in vitro inhibition binding constants (Ki) of ligands
1, 3 and 4 were determined using an established com-
petition assay (rat cortical SERT, [3H]paroxetine)30
which is a modi®ed method of Habert.31 The measured
Ki data (meanÆS.E.M., n=3) are summarized in Table
1. The in vitro assays were performed with aliquots of
Methyl groups were favored as initial test substituents
based on their synthetic accessibility and value in the
past to e€ectively probe SAR trends in the SSRI par-
oxetine drug family.19 Furthermore, preliminary data
acquired with methyl-substituted analogues could guide
the subsequent study and development of related ¯uo-
rine atom or methoxyl group appended derivatives (20-
¯uoromethyl and 20-methoxymethyl, respectively; struc-
tures not shown) of 3 and 4 as potential PET ligands.
Other investigations have revealed that decreased SERT
binding potency can be observed by placing uncon-
strained alkyl groups at or near the terminal amine end
of SERT inhibitors.16,19 Therefore, it was thought that
target ligand 4 would serve to further de®ne the sensi-
tivity of this pharmacophore region to simple sub-
stituents. Described below are the racemic syntheses of
ligands 3 and 4 and the assessment of their in vitro
binding potency utilizing rat cortical SERT and
[3H]paroxetine.
Table 1. The in vitro inhibition binding constants (Ki) and equipo-
tent molar ratios (EPMR) of the standard 6-nitroquipazine (1) and the
target analogues (3±4)
Ligand
Inhibition constant, Ki (nM)a
EPMRb
1
3
4
0.163Æ0.053
0.081Æ0.061
4.56Æ2.4
1.0
0.49
28
aData from competition assays using rat cortical membranes labeled
with [3H]paroxetine. The Ki data calculated from IC50 values represent
the meanÆS.E.M. of three or more individual determinations.
bThe equipotent molar ratio (EMPR) is the ratio of Ki values; Ki ana-
logue (3 or 4): Ki 6-nitroquipazine (1, standard).
Scheme 1. Synthetic routes to the target ligands 3 and 4.

J. M. Gerdes et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2643±2646
2645
partially puri®ed rat cortex membrane tissue (Pel-Freeze
Biologicals, Inc.). Suspensions were incubated with
0.25 nM [3H]paroxetine (DuPont-NEN, sp. act. 2.5 Ci
found to be of comparable potency relative to the
known high anity SERT selective agent 5-iodo-6-
nitroquipazine, ligand 2.
1
6
mmol
)
and decreasing concentrations (10
to
10 11 M) of 1 (standard; Research Biochemicals, Inc.),
3, 4 or unlabeled paroxetine (SmithKline-Beecham).
Incubations were terminated by dilution with ice-cold
bu€er and then ®ltered and washed rapidly on a Bran-
dell cell harvester, air dried and then counted with a
Packard scintillation counter.
Since placement of a methyl group at the 20-position of
1 serves to enhance SERT anity, then it is plausible
that certain substituents at this location may be inter-
acting with a portion of the SERT binding domain that
normally accommodates the second aromatic ring moi-
ety found in most SSRIs. The reduced binding potency
of ligand 4 suggests that the 30-position of the parent
drug 1 is a location that is sensitive to substitution. This
observation parallels an earlier ligand trend, where
placement of simple alkyl groups at or near the terminal
amine end of some SERT inhibitors results in decreased
SERT anity.
Speci®c binding was de®ned as the binding di€erence in
the presence and absence of unlabeled paroxetine. Non-
speci®c binding was de®ned by 1 mM paroxetine to the
incubation media. Binding data were analyzed by con-
ventional Hill plots (data not shown) where percent of
total speci®c binding versus log molar concentration
of inhibitor 1, 3, 4 or unlabeled paroxetine (six or more
data points per curve) were plotted and the inhibitor
concentrations (IC50) required to obtain 50% inhibition
of [3H]paroxetine binding were determined. Assay data
points were accomplished in triplicate and the experi-
ments were repeated three times on di€erent days. The
inhibition binding constants (Ki) were calculated from
the established relationship32 Ki=IC50/(1+[L]/KD)
where [L]=is the concentration of free (unbound)
[3H]paroxetine and KD=0.15 nM.31
The racemic 20-methyl-6-nitroquipazine, ligand 3, is a
potent SERT inhibitor that serves as a new lead agent
for further study. Investigations to improve the syn-
thetic yield of 3 and to assess the eudesmic ratio of the
enantiomers of 3 are in progress. E€orts to prepare the
20-¯uoromethyl- and 20-methoxymethyl-analogues of 1
as non-radioactive congeners of prospective PET agents
and also experiments to demonstrate SERT selectivity
of ligand 3 and variants are underway. The results from
these SARs and related studies shall be reported in the
near future.
As shown in Table 1, the measured Ki values (nM)
indicate that ligand 3 is more potent than the parent
agent 1, whereas ligand 4 is less potent than 1 for dis-
placing [3H]paroxetine from rat cortical SERT. Ligand
3 was 50 times more potent than 4 in the competition
assay. The equipotent molar ratios (EPMR) calculated
for 3 and 4 (Ki of 3 or 4:Ki of 1) are 0.49 and 28,
respectively. A similar assay carried out earlier by
others16,33 evaluating 5-iodo-6-nitroquipazine 2 and the
parent ligand 1 provided an EMPR value of 0.83 (Ki of
2:Ki of 1=0.19 nM:0.23 nM). A comparison of the
EPMR values for ligands 2 versus 3 reveals that the
20-methyl-6-nitroquipazine 3 is of similar potency to the
5-iodo variant 2.
Acknowledgements
This research was supported by grants NIH NS36405,
Research Corporation, M. J. Murdock Trust and CWU
Grants Program (JMG, SCD and PAW) and by the
Oce of Biological and Environmental Research, US
Department of Energy, under contract No. DE-AC03-
76SF00098 (SET). We are grateful to Mr. Marc Walker
for his technical assistance.
References and Notes
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(2H, m), 3.09 (1H, t, J=12.2 Hz), 3.18 (1H, d, J=12.1 Hz),
4.52 (2H, d, J=12.5 Hz), 7.07 (1H, d, J=9.0 Hz), 7.66 (1H, d,
J=9.5 Hz), 7.96 (1H, d, J=9.5 Hz), 8.30 (1H, dd, J=9.0 Hz,
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20. Yields reported are of isolated puri®ed products and are
not optimized.
21. All synthetic intermediates and ®nal products were puri®ed
by column chromatography (silica gel 60, isopropylamine:ethyl
acetate/hexane, 1:1.5:7.5) with the exception of amine 6 which
was recrystallized from ether/hexane, 1:9 (mp 137±139 ꢀC).
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33. During the time the binding assessments were performed,
ligand 2 was unavailable and, thus, the established EPMR
method of evaluation (ref 16) was used to compare ligand
potency (2 vs 3) from experiments performed at di€erent times
and in separate laboratories.