NO-SSRIs: Nitric oxide chimera drugs incorporating a selective serotonin reuptake inhibitor

Article abstract of DOI:10.1021/ml2000033

Hybrid nitrate drugs have been reported to provide NO bioactivity to ameliorate side effects or to provide ancillary therapeutic activity. Hybrid nitrate selective serotonin reuptake inhibitors (NO-SSRIs) were prepared to improve the therapeutic profile of this drug class. A synthetic strategy for use of a thiocarbamate linker was developed, which in the case of NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4 within 7 h. In cell culture, NO-SSRIs were weak inhibitors of the serotonin transporter; however, in the forced swimming task (FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an NO-chimera nitrate developed for Alzheimer's disease (GT-1061) were made in the step through passive avoidance (STPA) test of learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential for NO-SSRIs to overcome the lag in onset of therapeutic action and provide cotherapy of neuropathologies concomitant with depression.

Full text of DOI:10.1021/ml2000033

LETTER
pubs.acs.org/acsmedchemlett
NO-SSRIs: Nitric Oxide Chimera Drugs Incorporating a Selective
Serotonin Reuptake Inhibitor
Samer Abdul-Hay, Isaac T. Schiefer, R. Esala P. Chandrasena, Min Li, Ramy Abdelhamid, Yue-Ting Wang,
Ehsan Tavassoli, Bradley Michalsen, Rezene T. Asghodom, Jia Luo, and Gregory R. J. Thatcher*
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood
Street, Chicago, Illinois 60612-7231, United States
S Supporting Information
b
ABSTRACT: Hybrid nitrate drugs have been reported to
provide NO bioactivity to ameliorate side effects or to provide
ancillary therapeutic activity. Hybrid nitrate selective serotonin
reuptake inhibitors (NO-SSRIs) were prepared to improve the
therapeutic profile of this drug class. A synthetic strategy for use
of a thiocarbamate linker was developed, which in the case of
NO-fluoxetine facilitated hydrolysis to fluoxetine at pH 7.4
within 7 h. In cell culture, NO-SSRIs were weak inhibitors of the
serotonin transporter; however, in the forced swimming task
(FST) in rats, NO-fluoxetine demonstrated classical antidepressant activity. Comparison of NO-fluoxetine, with fluoxetine, and an
NO-chimera nitrate developed for Alzheimer's disease (GT-1061) were made in the step through passive avoidance (STPA) test of
learning and memory in rats treated with scopolamine as an amnesic agent. Fluoxetine was inactive, whereas NO-fluoxetine and GT-
1061 both restored long-term memory. GT-1061 also produced antidepressant behavior in FST. These data support the potential
for NO-SSRIs to overcome the lag in onset of therapeutic action and provide cotherapy of neuropathologies concomitant with
depression.
KEYWORDS: SSRI, nitric oxide, antidepressant, cognition, hybrid drug, nitrate
his paper is the first report on hybrid nitrate selective
serotonin reuptake inhibitors (nitrooxy-SSRIs or NO-SSRIs;
the modified forced swim test (FST), a model of “learned
helplessness” that is used extensively as an antidepressant
screen in industry.14 Criticisms of the FST include the immediate
induction of antidepressant activity in the FST by drugs that
clinically show a therapeutic lag of several weeks. Despite the
imperfections of the FST, antidepressant drugs almost universally
decrease the “immobility” time of the animal in FST; therefore, a
drug that is ineffective in the FST is unlikely to progress as a therapy
for neurological disorders in which depression is comorbid. Because
NOS inhibitors have been reported to decrease immobility in the
FST,15,16 it was essential to determine the influence on immobility
of NO-SSRIs.
A NO chimera that incorporates an antidepressant pharma-
cophore, for example, fluoxetine (2) or paroxetine (4), repre-
sents a novel antidepressant therapeutic strategy that might be of
general use in depression or of specialized use when cognition
enhancement is required or beneficial. The appropriate commer-
cially available SSRI drugs were modified at the secondary
nitrogen position via formation of a thiocarbamate bond. Pre-
liminary treatment of the parent SSRI molecules with phosgene
followed by mercaptoethanol (Scheme 1, route A) gave a mixture
of the desired thiocarbamate-containing compounds, either 2a or
4a, as well as approximately equimolar quantities of the
T
Figure 1). Akin to an approach taken for NO chimera hybrid drugs
in Alzheimer's disease (AD)1 and NO-donor NSAIDs for a variety
of indications,2,3 the NO bioactivity resulting from these drug
hybrids is proposed to enhance and ameliorate the actions of the
primary pharmacophore. (1) The clinical lag in onset of antide-
pressant action is a key source of noncompliance in SSRI pharma-
cotherapy, which is linked to the delay in elevation of brain-derived
neurotrophic factor (BDNF).4,5 NO is known to regulate BDNF
production via positive feedback, since BDNF itself induces neuro-
nal NO synthase (NOS) expression.6,7 (2) Hybrid nitrates and
nitrate NO-chimera drugs have been shown to reverse cognition
deficits in animal models of dementia and AD,1,8,9 and it has
been suggested that therapeutic strategies for AD should target
depression.10,11 Several theories have been postulated for the
comorbidity of depression and dementia, and patients with
lifetime depression have a 50% higher risk of developing AD
and more advanced AD pathophysiology.12,13
In pursuit of NO-SSRI hybrid drugs, two initial hurdles were
presented as follows: (1) to develop a general prodrug strategy
for amine SSRI drugs, since most hybrid nitrates reported rely on
linker modification of hydroxyl and carboxylate groups in the
drug itself, and (2) to ascertain whether the NO-SSRI retained
the antidepressant activity of the primary pharmacophore. To
determine the antidepressant activity of NO-fluoxetine, we used
Received: January 15, 2011
Accepted: July 25, 2011
Published: July 26, 2011
r
2011 American Chemical Society
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Figure 1. Hybrid nitrate NO chimeras and NO-SSRIs.
Scheme 1a
a Reagents and conditions. (i) Phosgene, TEA, CH2Cl2, 0 °C, 1 h. (ii) Mercaptoethanol, TEA, CH2Cl2, room temperature, 8 h. (iii) CDI, TEA, CH2Cl2,
room temperature, 8 h. (iv) CH3I, THF, room temperature, 12 h. (v) Mercaptoethanol, TEA, CH2Cl2, 0 °C, 8 h.
undesired carbamate derivatives, 2b and 4b, respectively. An
alternative synthetic approach was taken entailing the treatment
of the SSRI with CDI to give intermediates 2c or 4c (Scheme 1,
route B), followed by methylation using iodomethane, and
subsequent treatment with mercaptoethanol to preferentially
yield the SSRI thiocarbamates 2a and 4a, respectively.
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LETTER
Scheme 2a
a Reagents and conditions. (i) PPh3, CBr4, CHCl3, 0 °C. (ii) AgNO3, MeCN, 80 °C. (iii) C(O)Cl2, TEA, CH2Cl2, 0 °C, 4. (iv) t-BuLi, tert-buty
4-oxopiperidine-1-carboxylate. (v) CF3CO2H. (vi) (R)-oxiran-2-ylmethyl 3- nitrobenzenesulfonate.
Figure 2. (A) Efficacy of NO-SSRIs and metabolites as compared to fluoxetine. HEK 293 cells stably transfected with the human serotonin transporter
(SERT) were treated with fluoxetine (varied concentrations; inset figure) or test compounds (100 nM). SERT inhibition data were normalized to
fluoxetine (100 μM) as 100% inhibition of reuptake and vehicle control as 0%. The data show means and SEMs from three separate experiments. (B)
Hydrolysis of NO-fluoxetine to fluoxetine. Breakdown of NO-fluoxetine (200 μM) to fluoxetine measured by HPLC-UV in PBS (50 mM PBS; pH 7.0,
7.4, 8.0). The peak area of fluoxetine is expressed as a ratio against total product and substrate; data show means and SEMs. (C) Bioavailability of
fluoxetine after administration of NO-fluoxetine. Quantitation of fluoxetine (2) by LC-MS/MS using MRM analysis of the m/z transition 310 f 44 after
ip administration of NO-fluoxetine (3; 15 mg/kg ip) at two time points before sacrifice. Data (N = 2) show means and SDs.
Conversion of alcohols to nitrate was readily achieved in two
steps by use of triphenylphosphine and carbon tetrabromide,
followed by treatment with silver nitrate, yielding the mononitrate
NO-SSRIs, 3 and 5 (Scheme 2). This procedure represents a
general approach toward hybrid nitrate derivatives of CNS-active
amines, using a thiocarbamate linker. The method was adapted for
the synthesis of one example of a dinitrate NO-SSRI (7) using a
nitrate-mercaptan synthon reported in 2001.17 A further example of
a dinitrate NO-SSRI (12) was prepared, making use of a pharma-
cophore explored by Eli Lilly in hybrid SSRI/5HT1A antagonist
drugs designed to overcome antidepressant therapeutic lag.18
To determine the SSRI activity of NO-fluoxetine (3), NO-
paroxetine (5), and their potential metabolic denitration products,
2a and 4a, inhibition of serotonin reuptake was measured in human
HEK-293 cells stably transfected with the serotonin transporter
(SERT). The affinity of fluoxetine itself for SERT in this assay was
reflected by the EC50 = 2.7 nM (Figure 2A). At 100 nM, fluoxetine
efficacy was 81%, whereas at this concentration, the NO-SSRIs
tested showed <15% efficacy. Although the putative denitration
metabolite, 4a, appeared to show higher activity, the data demon-
strate that the hybrid prodrugs and metabolites are weak inhibitors
of SERT.
To determine the stability of the thiocarbamate linker selected
for these hybrid nitrate prodrugs, the lability of NO-fluoxetine
was assayed at three pH values spanning physiological pH
(Figure 2B). LC-MS/MS was used to determine that fluoxetine
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Scheme 3
was the sole hydrolysis product observed. The denitration
product 2a was not observed under these conditions, because
denitration requires reductive conditions or alkaline pH. The
fluoxetine concentration was determined using UV detection at
multiple time points, showing that the decomposition of NO-
fluoxetine reached completion at only 7 h, with a half-life at
physiological pH of approximately 100 min (Figure 2B). The
rate of hydrolysis increased with pH, compatible with a base-
catalyzed mechanism of thiocarbamate cleavage and consequent
decarboxylation (Scheme 3). Chemiluminescence detection of
NO from the sealed headspace above a mixture of NO-fluox-
etine (500 μM) in PBS-containing glutathione revealed produc-
tion of low amounts of NO (8.9 ( 1.9 nM) after 30 min of
reaction.
NO-SSRIs are designed as prodrugs; therefore, detection of
drug at the site of action was seen as important. Administration to
mice of 3 (15 mg/kg ip) 20 or 120 min prior to sacrifice, followed
by quantitation of 2 by LC-MS/MS, demonstrated bioactivation
of NO-fluoxetine to fluoxetine (2) in both plasma and brain at a
favorable brain/plasma ratio (Figure 2C).
In the FST, the rat is acclimatized to a water-filled glass
cylinder, in which the animal may display movements consis-
tent with struggle, swimming, and immobility. Immobility is
associated with the minimum movement required to maintain
the animal at the surface of the water. Antidepressant drugs
almost universally decrease the “immobility” time of the animal
in FST. In addition, this animal model is reported to respond
differently to serotonergic versus adrenergic and catecholami-
nergic drugs: Whereas all reduce immobility, serotonergic
drugs do not increase struggle behavior.14 NO-fluoxetine (3;
26.5 mg/kg equivalent to 20 mg/kg fluoxetine) was delivered in
three ip injections 1, 5, and 24 h before the test phase of FST, a
dose and delivery regime based upon literature reports for
fluoxetine.19 NO-fluoxetine was observed to reduce the im-
mobility time significantly in the FST relative to the vehicle
control and to increase swimming time significantly with no
significant effect on struggle time (Figure 3A). These charac-
teristics are compatible with antidepressant activity delivered by
serotonergic drugs.
Figure 3. (A and B) Antidepressant activity of NO-fluoxetine and GT-
1061. Effect of NO-fluoxetine (A) and GT-1061 (B) on the duration of
struggle, swimming, and immobility in the FST. Rats received ip
injections of drug or vehicle before testing. Data represent the number
of 5 s blocks of each type of behavioral activity presented as means and
SEMs (N = 6). (C) Pro-cognitive effects of NO-fluoxetine. The effect of
NO-fluoxetine on long-term memory retention in the step through
passive avoidance (STPA) task as compared to equimolar fluoxetine,
GT-1061, and the clinical nitrate vasodilator, ISDN. Memory was tested
48 h after training. Data show latency to as means and SEMs (N = 9À11;
for fluoxetine N = 7, since mice did not learn the task in training). The
data were evaluated by ANOVA, followed by Tukey's posthoc test: ns,
not significant; p > 0.05; *p < 0.05; **p < 0.01; and ***p < 0.001.
The positive antidepressant data obtained with NO-fluoxetine
dispelled the hypothesis, based upon reports on NOS inhibitors,15,20
that nitrate NO donors would de facto be pro-depressant in the
FST. It was of further interest to test in FST the NO chimera, GT-
1061, an experimental AD drug. An administration protocol was
used identical to that for NO-fluoxetine, but dosing at 10 mg/kg
(comparable to reported procognitive doses of this NO chimera8,21).
The nitrate GT-1061 was observed to display an “antidepressant
profile” comparable to that of NO-fluoxetine (Figure 3B).
Given the comparable activity of NO-fluoxetine and GT-1061
in the FST and the demonstrated ability of GT-1061 to reverse
cognition deficits in animal models using a variety of paradigms,
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the two drugs were compared in a simple animal model of
learning and memory. The step-through passive avoidance
(STPA) task was used to measure the reversal of a stimulus-
response spatial memory deficit induced by the amnesic agent
scopolamine that blocks muscarinic acetylcholine receptor acti-
vation. The STPA task tests learned aversion memory, relying on
the natural preference of rats for dark areas over brightly lit and
open areas. Following a period of habituation, the rat is trained.
The animal is placed in the light compartment of the apparatus
and inevitably crosses to the dark compartment of the apparatus
at which time an electrical shock is delivered. The animals rapidly
learn to avoid moving into the dark compartment. Retention of
this newly learned behavior can be tested on subsequent days to
assess the strength of the aversive memory, by measuring the
latency to enter the dark area. NO-fluoxetine and GT-1061 were
compared with fluoxetine and the clinical nitrate, isosorbide
dinitrate (ISDN) administered at equimolar doses (NO-fluoxetine,
2.05 mg/kg; GT-1061, 1.0 mg/kg; and fluoxetine, 1.54 mg/kg).
Long-term memory retention was measured 48 h after training in
the absence of drugs (Figure 3C). No significant effect was
apparent for fluoxetine relative to saline vehicle control. Inter-
estingly, 5 of 12 animals receiving fluoxetine failed to learn the
STPA task during the training phase, as compared to zero in the
NO-fluoxetine treatment group. Regardless of cause, NO-fluoxetine
significantly improved long-term memory, demonstrating that
the hybrid nitrate prodrug possesses biological activity in vivo
additional to that of the parent SSRI and in accord with
observations on GT-1061.8
(2 mM) in phosphate buffer at the appropriate pH was monitored by
reverse phase HPLC using UV detection at 227 nm to monitor
fluoxetine formation, which was quantified using peak area ratios with
respect to the total UV absorbance. NO-fluoxetine prodrug was
administered to mice 20 or 120 min prior to sacrifice and analysis by
LC-MS/MS.
Human embryonal kidney cells (HEK-293) stably transfected with
serotonin transporter (SERT) cDNA (a kind gift from Dr. Barker,
Purdue University) were used to determine the serotonin reuptake
inhibitory activity of test compounds.
The test was performed using male SpragueÀDawley rats weighing
200À250 g. Rats were trained to swim in the water tank 24 h prior to
testing for 15 min. Drug was administered by ip injections 24, 5, and 1 h
before the test at a dose of 26.5 mg/kg, which is equimolar to 20 mg/kg
fluoxetine. The 5 min test was divided into 60 blocks of 5 s each. The
major behavior in each 5 s block was assigned for the whole block by two
observers blinded to the treatment: climbing, swimming, or immobility.
The number of blocks representing each behavior was counted.
STPA was performed on male C57BL/6 mice of 8À10 weeks of age
and weighing 22À27 g. The avoidance experiment was divided into
three phases: habituation, training, and retention. The training phase,
performed 2 h after habituation, consisted of individually placing animals
in the illuminated compartment, with an electric shock (0.6 mA for 2 s)
delivered immediately after the mouse entered the dark compartment,
accompanied by the door closure. The time (latency) to enter the dark
chamber was recorded for each mouse. Trials were repeated for a
maximum of five trials or until the mouse remained in the light compart-
ment for 300 s in one trial. The retention test was conducted 48 h after
training, consisting of a single repeat of the training protocol without
foot shock. The retention trial ended when the mouse entered the dark
box or 300 s had elapsed. Scopolamine (1 mg/kg) dissolved in
physiological saline was administered by ip injection 30 min before
the training phase, and drugs dissolved in 25% DMSO were adminis-
tered by ip injection 20 min before the training.
Current guidelines for pharmacotherapy of depression pro-
pose SSRIs as a first line therapy; however, the 2À6 week
therapeutic lag before onset of behavioral improvement presents
an impediment to compliance and therapy. The neurotrophin
hypothesis posits a causative role for reduced activity of CREB
(cAMP response element binding protein) and BDNF in patho-
genesis of depression.22,23 Downstream of neurotransmitter
systems, neurotrophin-mediated long-term adaptation including
enhancement of neurogenesis and neuronal plasticity is thought
essential for clinical antidepressant efficacy and compatible with
the observed antidepressant therapeutic lag.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental proce-
b
dures, LC-MS/MS data, and characterization data. This material
is available free of charge via the Internet at http://pubs.acs.org.
Many drug classes would benefit from improvement of side
effect profiles, which is a key incentive for prodrug and hybrid
drug design. There are significant problems specifically asso-
ciated with antidepressant therapy that might be overcome by
new hybrid therapeutic agents and prodrugs that overcome the
lag in onset of therapeutic action and provide cotherapy of at least
one of the associated symptoms or neuropathologies concomitant
with depression. Enhancement of CREB/BDNF signaling repre-
sents a therapeutic target for such hybrid and prodrug strategies.
Activation of CREB/BDNF can be elicited by enhancement of
NO/cGMP signaling, and because organic nitrates are proven
clinically, hybrid nitrates represent a reasonable therapeutic
approach.1,24 A simple, general synthesis of thiocarbamate-linked
hybrid nitrates is described, yielding NO-SSRI prodrugs that
release the parent SSRI and NO bioactivity on bioactivation.
NO-fluoxetine retained the antidepressant activity of fluoxetine
and the procognitive actions of the NO chimera nitrate, GT-
1061, in animal behavioral models.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: thatcher@uic.edu.
Funding Sources
This study was funded in part by NIH R21 AT002299 and by the
Alzheimer's Drug Discovery Foundation.
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’ EXPERIMENTAL PROCEDURES
See the Supporting Information for specific reaction quantities, full
product characterization, and detailed methodologies. Reaction of 3
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