γ-aminobutyric acid amides of nortriptyline and fluoxetine display improved pain suppressing activity

Article abstract of DOI:10.1021/jm900143u

The GABA amides of the antidepressants nortriptyline and fluoxetine, 1 and 2, were compared to their respective parent compounds in rodent models of pain. The amides significantly reduced early nociceptive and late inflammatory responses compared to nortr

Full text of DOI:10.1021/jm900143u

3010
J. Med. Chem. 2009, 52, 3010–3017
γ-Aminobutyric Acid Amides of Nortriptyline and Fluoxetine Display Improved Pain
Suppressing Activity
Ada Rephaeli,*,† Irit Gil-Ad,‡ Ana Aharoni,§ Igor Tarasenko,† Nataly Tarasenko,† Yona Geffen,| Efrat Halbfinger,|
Yotam Nisemblat,| Abraham Weizman,‡ and Abraham Nudelman*,§
Laboratory of Pharmacology and Experimental Oncology and Laboratory of Biological Psychiatry, Felsenstein Medical Research Center,
Sackler Faculty of Medicine, Tel-AViV UniVersity, Ramat AViV, Israel, Department of Chemistry, Bar Ilan UniVersity, Ramat Gan, Israel, and
Bioline InnoVations Jerusalem LP, 19 Hartum Street, Jerusalem, Israel
ReceiVed February 4, 2009
The GABA amides of the antidepressants nortriptyline and fluoxetine, 1 and 2, were compared to their
respective parent compounds in rodent models of pain. The amides significantly reduced early nociceptive
and late inflammatory responses compared to nortriptyline or fluoxetine, where 1 exhibited overall better
efficacy than 2. Amide 1 was most efficacious in lowering cytokine secretion, edema and hyperalgesia
induced by formalin and λ-carrageenan, respectively. Thus, 1 is a promising candidate for the treatment of
pain.
Introduction
suggest that compounds with greater norepinephrine reuptake
inhibitory (NRI) activity versus serotonin reuptake inhibitory
(SRI) activity would be more effective in the treatment of pain
than compounds with only SRI activity.15
Inadequate management of postoperative and trauma pain,
cancer, and chronic noncancer pain is widely prevalent.1-4 Pain
can be divided into two main categories: acute and chronic.
Acute pain is a protective response to tissue injury or inflam-
mation and is most often nociceptive, namely, is caused by or
is a response to a tissue damaging stimulus. Chronic pain that
may be nociceptive or neuropathic results from neuronal
transmission of pain, either peripherally or in the central nervous
system (CNSa). The spinal column and the CNS are modulated
by excitatory and inhibitory neurotransmitters. Norepinephrine
and serotonin are inhibitors of pain transmission, whereas other
neurotransmitters, e.g., glutamate and N-methyl-D-aspartate are
excitatory.1,5,6 Antidepressant and antiepileptic drugs are thought
to relieve neuropathic pain through interaction with specific
neurotransmitters and ion channels.7,8 The tricyclic antidepres-
sants amitriptyline and its metabolite nortriptyline, which show
inhibition of norepinephrine and serotonin reuptake, were
reported to be efficacious in the treatment of neuropathic and
non-neuropathic pain syndromes.9-12 Serotonin inhibits the
transmission of pain at the spinal cord and midbrain level. The
efficacy of tricyclic antidepressants in the treatment of neuro-
pathic pain is evident at lower dosages than those typically used
to treat depression and is independent of their antidepressant
effects.13,14 Experimental models of pain and clinical data
γ-Aminobutyric acid (GABA) is the main inhibitory neu-
rotransmitter of the CNS, and its deficiency is responsible for
many pain states.5 Neuropathic lesions of the type that evoke
hyperalgesic states are also known to induce a loss of GABAer-
gic inhibition in the spinal dorsal horn.16,17 This phenomenon
could be explained by a decrease in dorsal horn levels of the
GABA synthesizing enzyme glutamic acid decarboxylase
(GAD) and associated neuronal apoptosis.18 Gabapentin and
pregabalin were initially synthesized to mimic the chemical
structure of GABA. However, they were found to act on voltage-
gated N-type calcium ion channels in the central nervous system
and not on the brain receptors of GABA.19
On the basis of the association between GABA deficiency
and pain events and the described analgesic activity of NRI and
SRI antidepressants, we synthesized new chemical entities
composed of GABA amides of nortriptyline 1 (BL-1021) and
of fluoxetine 2 (BL-1024)20 and tested them in rodent models.
The GABA amides of the antidepressants were originally
designed on the basis of the analogous GABA ester of the typical
antischizophrenic agent perphenazine (BL-1020), which we
reported earlier.21 This ester was found to behave as a mutual
prodrug that upon metabolic hydrolysis in the brain concomi-
tantly releases perphenazine and GABA. In the present case,
the GABA derivatives are amides. Although amides are
significantly more stable than esters in biofluids, it is difficult
to predict whether they will undergo metabolic hydrolysis or
be active as the intact molecules. Herein, we describe the
chemical and biological studies conducted with the GABA-
antidepressant amides.
* To whom correspondence should be addressed. For A.R.: (address)
Laboratory of Pharmacology and Experimental Oncology, Felsenstein
Medical Research Center, Tel-Aviv University, Rabin Medical Center Petah
Tikva 49100, Israel; (phone) +972 3 937 6126; (fax) +972 3 922 8096;
(e-mail) adarep@post.tau.ac.il. For A.N.: (address) Department of Chemistry,
Bar-Ilan University, Ramat Gan 52900, Israel; (phone/fax) +972-3-5318314;
(e-mail) nudelman@mail.biu.ac.il.
†
Laboratory of Pharmacology and Experimental Oncology, Tel-Aviv
University.
‡
Laboratory of Biological Psychiatry, Tel-Aviv University.
Bar Ilan University.
Bioline Innovations Jerusalem LP.
§
Results and Discussion
|
GABA amide of nortriptyline 1 and of fluoxetine 2 were
prepared as shown in Scheme 1.
a Abbreviations: CNS, central nervous system; DDW, doubly distilled
water; GAD, glutamic acid decarboxylase; GABA, γ-aminobutyric acid;
NRI, norepinephrine reuptake inhibitory; SRI, serotonin reuptake inhibitory;
PMSF, phenylmethylsulfonyl fluoride; eqM, equimolar; TNF-R, tumor
necrosis factor-R; INF-γ, interferon-γ; EDC, 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride; [3H]CGP, ([S-(R,R)]-[3-[[1-(3,4-dichlo-
rophenyl)ethyl]amino]-2-hydroxypropyl]-([3,4-3H]cyclohexylmethyl)phos-
phinic acid.
Effect of the Antidepressants Nortriptyline, Fluoxetine,
and Their GABA Amides 1 and 2 on Pain Sensation Using
the Hot Plate Test. Amides 1 and 2 were evaluated as
analgesics in comparison to their parent antidepressants on the
hot plate that measures the response to a noninflammatory acute
10.1021/jm900143u CCC: $40.75
2009 American Chemical Society
Published on Web 04/20/2009

GABA Amides of Nortriptyline and Fluoxetine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3011
Scheme 1. Synthesis of GABA Amidesa
a (a) Nortryptiline/Boc-GABA/EDC/Et3N ratio 1:1.2:1.2:1, CH2Cl2, room temp, 48 h; (b) fluoxetine/Boc-GABA/EDC/Et3N ratio 1:1.2:1.2:1, CH2Cl2,
room temp, 48 h; (c) 4 N HCl/EtOAc, room temp, 4 h.
nociceptive input used to study central antinociceptive activity.22
The latency of the withdrawal response evoked by exposing
mice paws to a thermal stimulus (hot plate set to 52 ( 0.2 °C)
determined the antinociceptive reaction. To assess the effect of
nortriptyline, fluoxetine, and amides 1 and 2 on pain perception,
mice were treated po with these drugs and their respective parent
antidepressants. Oral administration of 0.25-4.5 mg/kg nortri-
ptyline and equimolar (eqM) doses of 1 and 7.5-40 mg/kg of
fluoxetine and eqM doses of 2 showed that after 240 min the
activity of nortriptyline and 1 leveled off at 0.5 mg/kg and that
of fluoxetine and 2 at 10 mg/kg and they were attenuated
thereafter (Figure 1 A,C). Treatment with 0.25, 0.5, and 4.5
mg/kg nortriptyline or eqM doses of 1 led to a significant
antinociceptive activity compared to vehicle treated mice, where
compound 1 at 0.5 and 4.5 mg/kg doses imparted a significantly
greater efficacy than nortriptyline at the corresponding doses
(p < 0.05). Treatment with 7.5 mg/kg fluoxetine or an eqM
dose of 2 did not impart a significant inhibition on the response
time to heat sensation, while treatment with 10-40 mg/kg
fluoxetine or eqM doses of 2 exhibited a significant increase in
the response time compared to vehicle treated mice. Only 10
mg/kg eqM dose of 2 imparted a significant increase in the
latency of the response compared to fluoxetine. At higher doses
(eqM doses of 20 or 40 mg/kg fluoxetine) the activity of
respective eqM dose 2 was attenuated and was similar to that
of fluoxetine (Figure 1C). These results indicate that 1 is an
effective antinociceptive agent at low eqM doses of 0.25-4.5
mg/kg while the activity of 2 peaked at 10 mg/kg and subsided
at higher doses, demonstrating the advantage of the former
amide. A time course study of the response to heat of mice
treated po with 0.4 mg/kg nortriptyline, an eqM dose of 1, 10
mg/kg fluoxetine, or an eqM dose of 2 showed that the response
after 2-4 h following amide treatment was delayed significantly
compared to that of the corresponding antidepressants or vehicle
(Figure 1B,D). After 5 h the analgesic effect subsided.
To evaluate the effect of repeated drug treatment, mice were
treated daily, po for 12 days, with nortriptyline (0.2 mg/kg) or
an eqM dose of 1 and the effect on heat sensation was
determined as indicated (Figure 2A). From the first day, 3-5 h
after treatment, the latency time of the response to heat increased
significantly in mice treated with nortriptyline (#, p < 0.05) or
1 compared to vehicle treated mice. From days 3 to 12, 3-5 h
after treatment, 1 imparted a significantly better antinociceptive
effect than nortriptyline (*, p < 0.05). Moreover, from day 10,
even prior to the daily treatment, as well as 3-5 h after
treatment, a significantly longer antinociceptive activity was
noted in mice treated repeatedly with 1 compared to those treated
with nortriptyline (*, p < 0.05) or vehicle (#, p < 0.05),
indicating that repeated administration of 1 prolonged the
duration of the analgesic effect (Figure 2A). Repeated dosing
with 1 not only imparted longer lasting effects from days 10 to
12 but also significantly increased the latency time of the
responses on days 10 and 12 compared to the latency time it
induced on days 1-5 (@, p < 0.05). The effect of repeated
administration of fluoxetine (po 10 mg/kg) or an eqM dose of
2 on days 1-11 was also evaluated. The analgesic effect of 2
3-5 h after treatment, from day 1, was significantly greater
than that of fluoxetine (*, p < 0.05). This advantage was
maintained throughout the experiment except for one time point,
on day 7, 5 h after the daily treatment (Figure 2B). It is noted
that the extent of the delay in the response induced by 2 on day
11 prior to treatment was 2-fold longer than that induced on
days 1-9 prior to the treatment, indicating a prolonged effect
after repeated doses of 2. A comparison of the latency induced
by 1 on days 10 and 12 to that induced by 2 on day 11 shows
that 1 induced longer latency period prior and 3-5 h after
treatment; therefore, 1 is superior to 2.
Chronic administration of GABA did not contribute to the
antinociceptive or cumulative analgesic effects. Mice treated
for 5 consecutive days with three oral doses of GABA (0.05,

3012 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Rephaeli et al.
Figure 1. Effect of heat sensation on mice treated with nortriptyline, fluoxetine, and their GABA amides: dose response and time course. The
latency of the reaction to heat (in s) using a hot plate set to 52 ( 0.2 °C was examined with male Balb-c mice (6/group). Dose-response of the
animals after 240 min of po treatments with vehicle or the indicated doses of nortriptyline and fluoxetine or eqM doses of their respective amide
1 or 2 are shown (A, C). Time course of the heat response of mice treated with 0.4 mg/kg nortriptyline, 10 mg/kg fluoxetine, and eqM doses of their
amides was determined (B, D). Results are expressed as the mean ( SEM: (*) p < 0.05, significant vs nortriptyline (A, B) and vs fluoxetine (C,
D); (#) p < 0.05, significant vs control (vehicle treated).
0.1, or 2.39 mg/kg) that corresponded to or exceeded the relative
amounts of GABA in 1 or corresponded to the eqM dose of
GABA in the efficacious dose of 2 did not exhibit a change in
the response to heat (data not shown).
treated with nortriptyline and 1, pain response was significantly
(p < 0.01) inhibited in both phases compared to vehicle treated
mice. In addition, 1, in the two treatment doses and phases,
was significantly (p < 0.05) more efficacious than the corre-
sponding doses of nortriptyline (Figure 3A,B). In mice treated
with fluoxetine, a significant reduction in the pain response in
the neurogenic phase was observed only at 30 mg/kg compared
to vehicle treated ones. However, treatment of mice with 10
and 30 mg/kg eqM doses of 2 imparted a significant reduction
of pain responses compared to fluoxetine or vehicle. In the
second inflammatory phase, 10 mg/kg fluoxetine and an eqM
dose of 2 were equally effective in reducing the response; 30
mg/kg fluoxetine further attenuated it, but an eqM dose of 2
imparted a significantly greater inhibition than fluoxetine (Figure
3C,D). Taken together, these results indicate that 1 is effective
at an over 20-fold lower dose than 2 and in both phases it
exhibited a superior antinociceptive activity compared to
nortriptyline.
Effect of Nortriptyline, Fluoxetine, and Amides 1 and 2
in the Formalin Pain Model. A second model acceptable for
nociception is the formalin induced pain test, characterized by
two phases. The first phase is brief and can be detected
immediately after formalin injection (0-5 min). The nociceptive
response in this phase results from a direct activation of
nociceptors and is similar to that of the hot plate test.22 The
second phase can be measured 20-60 min after formalin
injection and is attributed mainly to the development of an
inflammatory reaction at the injection site as well as to increased
synaptic transmission in the spinal cord. Animals treated with
formalin exhibited a typical flinching behavior of the injected
paw. In comparison to the hot plate test, which is indicative of
acute pain, the formalin test can also be used to predict the
potential of analgesic agents for the treatment of chronic pain
due to inflammation.23
Balb-c mice were treated 2 h prior to the formalin injection
with nortriptyline (0.5 and 5 mg/kg), fluoxetine (10 and 30 mg/
kg), and eqM doses of 1 and 2. The mice in the control group
were treated with the vehicle. Mice were then placed in an
acrylic observation chamber, and the frequency of pain-related
flinching was recorded during the early (0-5 min) and the late
(25-35 min) phases following the injection of formalin. In mice
Effect of Nortriptyline, Fluoxetine, and Their GABA
Amides 1 and 2 on Sedation and Anxiety. Since it is important
that analgesics should exert minimal sedation and anxiety, the
effect of 1 and 2 on the behavior of mice in the open-field test
was evaluated. This test is an acceptable model for psychiatric
disorders, involving confrontation of rodents with an anxiety
situation by a transfer to a new, large open field.24 In addition
to assessing anxiety, the test measures the parameters of motor
activity and sedation (immobility). Table 1 shows the effect of

GABA Amides of Nortriptyline and Fluoxetine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3013
Figure 2. Effect of daily administration of nortriptyline, fluoxetine, and their GABA amides on latency of the nociceptive reaction to heat. Balb-c
mice were treated daily with 0.2 mg/kg nortriptyline (n ) 6), an eqM dose of 1 (n ) 6), 10 mg/kg fluoxetine (n ) 10), or an eqM dose of 2 (n )
10) at the indicated days of treatment, and their latency of reaction to heat (s) on the hot plate (52 ( 0.2 °C) was measured prior to treatment (time
0) and 3 and 5 h after treatment. Results are expressed as the mean ( SEM: (#) p < 0.05 significant vs control; (*) significant vs eqM nortriptyline
or fluoxetine; (@) p < 0.05 significant vs previous days.
nortriptyline (1, 5, and 15 mg/kg), fluoxetine (5, 10, and 20
mg/kg) and eqM doses of 1 and 2 on distance moved, velocity,
and sedation in the open field on mice treated po with the drugs
compared to those treated with vehicle. Nortriptyline at 1 and
5 mg/kg doses did not modify significantly the distance moved
or velocity, while at 15 mg/kg a significant decrease was found
in both parameters (p < 0.05 vs controls). At the same time, 1
at 15 mg/kg eqM dose did not significantly affect these
parameters. At this dose a tendency toward increased immobility
(more sedation) compared to control was observed in animals
treated with both nortriptyline and 1; however, the difference
did not reach statistical significance. Mice treated with high
doses of fluoxetine (20 mg/kg) and an eqM dose of 2 exhibited
a significant (p < 0.01) reduction in distance moved and
velocity. The immobility observed at these doses increased
significantly with fluoxetine (p < 0.05), and a tendency (p >
0.05) to immobility was observed with 2. These results show
that fluoxetine at a high dose (20 mg/kg) significantly reduced
distance moved and velocity and increased sedation, whereas
an eqM dose of 2 decreased distance moved and velocity without
affecting immobility. Compound 1 at 15- to 30-fold higher doses
than its effective antinociceptive doses did not affect distance
moved, velocity, and immobility, while 2 reduced motor activity
at about 2-fold of its effective pain suppressing dose. Therefore,
a greater dose range of 1 can be used without imparting adverse
effects, and its higher analgesic activity cannot be attributed to
sedation.
Effect of Nortriptyline and Its Amide 1 on Inflamma-
tion. Inflammatory cytokines have been widely implicated in
both the establishment and the perpetuation of the inflammatory
process and neuropathic pain-related behavior in rodents.25 The
efficacy of 1 in the second phase of the formalin test led us to
investigate its effect on cytokine secretion at the formalin-
injection site and its anti-inflammatory activity against λ-car-
rageenan-induced rat paw edema.
The tissue from the formalin injected site was analyzed after
24 h for the level of the proinflammatory cytokines interferon-γ
(INF-γ) and tumor necrosis factor-R (TNF-R). Nortriptyline (po,
0.5 mg/kg), an eqM dose of 1, gabapentin (ip, 50 mg/kg), and
a mixture of nortriptyline (po, 0.5 mg/kg) and an eqM po dose
of GABA significantly reduced the TNF-R levels compared to
those observed in vehicle treated mice. However, 1 imparted a
significantly (p < 0.05) greater reduction compared to nortrip-
tyline, gabapentin, or the mixture of nortriptyline and GABA
(Figure 4A). The levels of INF-γ were significantly (<0.02)
reduced only in mice treated with 1 (Figure 4B). These results

3014 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Rephaeli et al.
Figure 3. Effect of nortriptyline, fluoxetine, and their amides on neurogenic and inflammatory response to formalin. Balb/c mice (8/group) were
treated with control vehicle, 0.5 or 5 mg/kg nortriptyline, and eqM doses of 1 at 2 h prior to 1% formalin (20 µL) injection to the right hind paw
(A, B). Similarly, Balb/c mice (5/group) were treated with control vehicle, fluoxetine 10 or 30 mg/kg, and eqM doses of 2 (C, D). Following the
formalin injection, the animals were placed in an acrylic observation chamber, and the number of times that the mice licked, bit, or shook the
injected paw was recorded as a quantitative indication of nociception. The early neurogenic phase (nociceptive response) was measured between
0 and 5 min (A, C), and the late phase (B, D) was measured between 25 and 35 min after the formalin injection. Results are expressed as the mean
( SEM: (#) p < 0.05, significant vs vehicle control; (*) p < 0.05, significant vs nortriptyline (A, B) and vs fluoxetine (C, D).
factors, e.g., prostaglandins.25 Wistar rats were treated po with
vehicle, 5 mg/kg nortriptyline, an eqM amount of 1, or 50 mg/
Table 1. Effect of Nortriptyline and Fluoxetine and Their GABA
Amides 1 and 2 on Distance Moved, Velocity, and Immobility on Male
Balb/c Mice in an Open Field Testa
kg of gabapentin administered ip. Two hours later the rats were
dose
(mg/kg)
distance moved
(cm)
velocity
(cm/s)
injected a solution of λ-carrageenan to the surface of their hind
paw. The volume of the injected site was measured 4 and 24 h
later, and the anti-inflammatory activity was expressed as the
change in volume between the two measurements (Figure 5A).
While nortriptyline did not reduce the edema, treatment with
gabapentin or 1 led to a significant edema volume reduction
compared to vehicle treatment (p < 0.01) and 1 to nortriptyline
(p < 0.05). The mean increase in edema volume in rats treated
with gabapentin was greater than that of rats treated with 1 (99
( 42 vs 32.2 ( 23, respectively); however, this difference was
not statistically significant.
drug
vehicle
nortriptyline
nortriptyline
nortriptyline 15
1
immobility (s)
8485 ( 610
8474 ( 407
7385 ( 447
6815 ( 51*
8053 ( 379
8157 ( 375
7413 ( 860
7485 ( 506
7970 ( 413
7.07 ( 0.51
7.13 ( 0.37
6.17 ( 0.38
5.68 ( 0.38*
6.72 ( 0.32
6.85 ( 0.30
6.18 ( 0.72
6.24 ( 0.42
6.64 ( 0.37
458.4 ( 41.0
468.6 ( 37.0
496.0 ( 45.4
547.9 ( 47.4
447.2 ( 16.7
467.9 ( 21.1
572.8 ( 86.7
552.2 ( 44.7
558.0 ( 42.0
1
5
eqM 1
1
1
eqM 5
eqM 15
5
10
20
fluoxetine
fluoxetine
fluoxetine
5783 ( 43.3** 4.78 ( 0.36** 709.3 ( 42.9*
2
2
2
eqM 5
eqM 10
eqM 20
7767 ( 371
8003 ( 589
6462 ( 506**
6.47 ( 0.31
6.67 ( 0.49
501.5 ( 21.6
536.3 ( 32.0
5.39 ( 0.42** 639.9 ( 50.5
a Treatment vs control: (*) p < 0.05; (**) p < 0.01. Results are expressed
as the mean ( SEM of six to eight determinations. There were six mice in
the groups treated with nortriptyline or 1 and eight mice in the groups treated
with fluoxetine or 2.
The local production of the inflammatory mediators activates
peripheral afferent fibers and leads to hyperalgesia and allodynia
manifested by increased sensitivity to thermal and mechanical
stimuli. To evaluate the effect of 1 on the heat sensation
following the λ-carrageenan injection, the rats were tested on
the hot plate. In rats treated with nortriptyline, an eqM dose of
1, or gabapentin (50 mg/kg), a significant increase in the latency
of the response 2-4 h after λ-carrageenan injection was
observed (Figure 5B). Consistent with the superiority of 1 in
the edema test, it also induced a significantly greater latency
than nortriptyline or gabapentin 4 h after λ-carrageenan injection,
demonstrating a significant inhibition of hyperalgesia in this test.
substantiate the observations obtained in the second phase of
the formalin test and suggest the potential of 1 in alleviating
pain induced by chronic inflammation.
The rat paw edema test, which measures the ability of a
compound to reduce local edema induced upon injection of the
irritant λ-carrageenan, is used for screening anti-inflammatory
agents. The edema development depends on the participation
of polymorphonuclear leucocytes with their pro-inflammatory

GABA Amides of Nortriptyline and Fluoxetine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3015
Figure 5. Compound 1 attenuated λ-carrageenan-induced paw edema:
(#) significant vs control; (*) significant vs nortriptyline and gabapentin.
Wistar rats (groups of 6) were treated po with vehicle, 5 mg/kg
nortriptyline, an eqM amount of 1, and ip with 50 mg/kg gabapentin.
After 2 h, paw edema was induced by injecting 100 µL of a solution
of 2.5% λ-carrageenan into the plantar surface of the left hind paws of
the rats. The area and the height of the induced edema were measured
4 and 24 h later using a caliper. The change in the volume of the injected
site from the first measurement at 4 h, the second measurement at 24 h,
after λ-carrageenan administration, for the different treatment groups
is shown in (A). The latency of the reaction of the rats to heat using
the hot plate set to 52 ( 0.2 °C was tested at 0, 2, and 4 h after
λ-carrageenan injection (B).
Figure 4. Compound 1 significantly reduced the level of interferon-γ
and TNF-R at the formalin-injection site: (#) significant vs control; (*)
significant vs all. Male Balb-c mice (8/group) were treated orally (po)
as indicated 120 min prior to the injection of formalin (20 µL of 1%
formalin solution) to their right hind paws. The treatment groups were
vehicle-treated control (po), 0.5 mg/kg nortriptyline and an eqM dose
of 1 (po), 50 mg/kg gabapentin (ip), and a mixture of nortriptyline 0.5
mg/kg and an eqM dose of GABA (po). After 24 h (after the injection
of formalin) the animals were sacrificed by CO2 inhalation and the
tissues from the injected site were removed, homogenized in 300 µL
of ice-cold PBS containing 0.4 mM NaCl, 0.05% Tween-20, 0.1 mM
phenylmethylsufonyl fluoride, and cocktail protease inhibitors. The
homogenate was centrifuged at 10000g for 30 min at 4 °C, the
supernatant was removed, and the protein content was determined and
assayed for TNF-R and interferon-γ using commercial ELISA kits
according to the manufacturer’s instructions.
pound 1 was active at 20-fold lower doses than 2 without
inducing sedation and reduced acute nociceptive sensation and
chronic inflammatory reaction. Thus, 1 has a potential of
alleviating acute and chronic pain.
In plasma samples spiked with 1, GABA was not detected
as a metabolite (data not shown). In an in vitro receptor binding
assay (performed by Cerep, France), the binding of an excess
of 1 (10 µM) to the GABAA, GABAB, and GABA-nonselective
GABA receptors resulted in 11%, -27%, and 6% inhibition of
control specific binding, respectively. Since the % inhibition
was lower than 50%, the Ki values were not determined. Thus,
it can be concluded that the in vitro binding affinity of 1 to
GABA receptors is low. These findings are similar to those of
the GABAergic drugs gabapentin and pregabalin that were
initially synthesized as GABA mimetics, and their mechanism
of action was found to be mediated through various mechanisms
not directly involving the GABA receptors.26 Since GABA is
not a metabolite of 1 and no specific binding to the GABA
receptors was observed, it is unlikely that GABA plays a part
in the mechanism of action. It is thus proposed that 1 is a new
chemical entity with analgesic activity superior to that of its
constituents nortriptyline and GABA. The mechanism by which
1 elicits its analgesic activity is under investigation.
Experimental Section
Chemistry. General Procedures. 1H and 13C NMR spectra were
obtained on Bruker AC 200 and DPX 300 MHz spectrometers.
Chemical shifts are expressed in ppm downfield from Me4Si (TMS)
used as internal standard. The values are given in δ scale. Multiplets
in quotation marks have second order characteristics. Low resolution
mass spectra (LRMS) were obtained in a QTof microspectrometer
in electrospray mode, and relative intensities of the measured peaks
appear in parentheses indicated as %. High resolution mass spectra
(HRMS) were obtained on an AutoSpec Premier (Waters, U.K.)
spectrometer in CI ()chemical ionization), CH4, mode. Progress
of the reactions was monitored by TLC on silica gel (Merck, article
5554). The purity of the compounds was evaluated by HPLC, and
compounds 1 and 2 were confirmed to be of g95% purity. The
HPLC (Agilent) system was used with a reverse phase Gemini 5
µm, 110 Å, 250 mm × 4.6 mm column at a flow rate of 1 mL/
min, using two different solvent systems. Solvent system I was
0.1% formic acid in water and acetonitrile. Solvent system II was
0.3% TFA in water (A) and 0.3% TFA in acetonitrile (B).
Commercially available compounds were used without further
purification.
Conclusion
Amides 1 and 2 in animal models of pain exhibited
significantly greater antinociceptive activity than their corre-
sponding parent compounds nortriptyline and fluoxetine. Com-
General Procedure I. To a solution of fluoxetine or nortriptyline
(1 equiv) and 4-tert-butoxycarbonylaminobutyric acid (1.2 equiv)
in anhydrous CH2Cl2, under N2, were added 1-(3-dimethylamino-

3016 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Rephaeli et al.
propyl)-3-ethylcarbodiimide hydrochloride (EDC) (1.2 equiv) and
Et3N (1 equiv). The mixture was stirred at room temperature for
48 h and then was washed with 1 N HCl (×3), 5% aqueous
NaHCO3 (×3), brine (×3), dried with MgSO4, filtered, and
evaporated to give the desired amide as a mixture of two rotamers.
Removal of N-Boc Group. General Procedure II. A solution
of 4 N HCl in EtOAc was added to a solution of an N-Boc protected
compound in EtOAc. The mixture was stirred for 4 h at room
temperature. Evaporation of the solvent gave the crude product
which was crystallized from methanol-ether.
(300 MHz, CDCl3) ppm δ 25.2/25.5 (CH2CH2CH2, major/minor),
28.5 (Me3C), 30.2/30.9 (CH2CO, minor/major), 33.6/36.0 (NMe,
minor/major), 36.4/37.5 (CHCH2CH2, major/minor), 40.5 (CH2NH),
45.4/46.3 (CH2NMe, major/minor), 77.4/78.4 (OCH, minor/major),
79.2 (CMe3), 115.8 (2CH, Ar), 123.6 (CCF3), 125.5 (CF3), 125.7/
125.8 (2CH, Ar, minor/major), 126.9 (2CH, Ar), 128.0/128.4 (CH,
Ar, major/minor), 128.9/129.2 (2CH, Ar, major/minor), 140.8/143.3
(C, Ar, major/minor), 156.2 (NHCO2), 160.4 (C, Ar), 172.6
(MeNCO). MS (ES+): m/z 517 (MNa+, 40%), 495 (MH+, 56%),
395 (MH+ - C5H8O2, 100%). HRMS: calcd for C26H34N2O4F3 (M+,
DCI/CH4) 495.2507, found 495.2471. HPLC of the products, using
solvent system II, indicated that the purity of 2 was 97.7% (retention
time, 19.7 min) with a minor impurity of 2.3% (retention time,
14.9 min). The N-Boc protective group of this compound was
removed as described in general procedure II to give 2 in
quantitative yield. The NMR spectra of the compound indicated
the presence of two rotamers in an approximate 3:2 ratio. 1H NMR
(300 MHz, MeOD) ppm δ 1.90 (“quint”, J ) 7 Hz, 2H,
CH2CH2CH2), 2.01-2.33 (m, 2H, CHCH2CH2), 2.46-2.60 (m, 2H,
CH2CO), 2.85-3.01 (m,2H, CH2NMe), 2.93/3.04 (s, 3H, NMe,
minor/major), 3.43-3.73 (m, 2H, CH2NH), 5.37/5.46 (dd, J ) 8,
4 Hz, 1H, OCH, major/minor), 7.02 (“t”, J ) 8.5 Hz, 2H, Ar),
7.2-7.52 (m, 7H, Ar). 13C NMR (300 MHz, MeOD) ppm δ 23.7/
23.9 (CH2CH2CH2, major/minor), 30.9/31.5 (CH2CO, minor/major),
34.0/36.4 (NCH3, minor/major), 37.0/37.8 (CHCH2CH2, major/
minor), 40.4 (CH2NH), 46.4/47.4 (CH2NMe, major/minor), 78.5/
79.2 (OCH, minor/major), 117.2 (2CH, Ar), 123.4 (CCF3), 124.0
(CF3), 127.1 (2CH, Ar), 127.7 (2CH, Ar), 128.9/129.1 (CH, Ar,
major/minor), 129.7/129.9 (2CH, Ar, major/minor), 141.7/142.1 (C,
Ar, minor/major), 161.7/161.9 (C, Ar, minor/major), 173.9/174.1
(NMeCO, minor/major). MS (ES+): m/z 395 (MH+). HRMS: calcd
for C21H25F3N2O2 ([MH+], DCI/CH4) 395.1946, found 395.1906.
Anal. Calcd for C21H25F3N2O2 ·HCl·H2O: C 56.19, H 6.29, N 6.24.
Found C 56.45, H 6.82, N 5.96.
10,11-Dihydro-5-(3-methylamonipropylidene)-5H-dibenzo-
[1,4]cycloheptene 4-amino-N-butaneamide Hydrochloride, 1.
tert-Butyl 3-(10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5-yllidene)-
N-methylcarbamoyl)propylcarbamate (N-tert-Boc-1), prepared as
described in the general procedure I from nortriptyline, was obtained
as a colorless oil (yield 81%) and was used as such without further
purification. Its NMR spectrum indicated the presence of two
rotamers in an approximate 1:1 ratio. 1H NMR (300 MHz, CDCl3)
ppm δ 1.43 (s, 9H, CMe3), 1.71/1.79 (“quint”, J ) 7 Hz, 2H,
CH2CH2CH2), 2.22-2.50 (m, 4H, CH2CO2, CHCH2CH2), 2.80/2.84
(s, 3H, NCH3), 3.06 (t, J ) 6.5 Hz, 2H, Ar-CH2CH2-Ar), 3.13 (t,
J ) 6.5 Hz, 2H, Ar-CH2CH2-Ar), 3.28/3.45 (“t”, J ) 7 Hz, 4H,
CH2NH, CH2NMe), 5.75-5.88 (m, 1H, CH), 6.98-7.29 (m, 8H,
Ar). 13C NMR (300 MHz, CDCl3) ppm δ 25.2/25.3 (CH2CH2CH2),
27.5/28.5 (CHCH2CH2), 28.4 (Me3C), 30.0/30.8 (CH2CO2), 31.9/
33.7 (Ar-CH2CH2-Ar), 32.0/33.7 (Ar-CH2CH2-Ar), 33.5/35.3
(NCH3), 40.4 (CH2NHCO2), 47.4/49.4 (CH2NMe), 78.7 (CMe3),
125.8 (CH), 125.9 (CH), 126.1 (CH), 126.2 (CH), 127.1 (CH), 127.4
(CH), 127.5 (CH), 127.8 (2CH), 128.0 (CH), 128.1 (CH), 128.2
(CH), 128.3 (CH), 128.6 (CH), 130.0/130.2 (CH), 136.9/137.0 (C,
Ar), 139.3/139.4 (C, Ar), 139.5/139.8 (C, Ar), 140.46/141.0 (C,
Ar), 144.4/146.1 (CdCH), 156.0 (HNCO2), 172.2 (MeNCO). MS
(ES+): m/z 449 (MH+, 81%), 349 (MH+ - C5H8O2, 100%).
HRMS: calcd for C28H37N2O3 (M+, DCI/CH4) 449.2823, found
449.2804. The N-Boc protective group of this compound was
removed as described in general procedure II to give 1 in
quantitative yield. The product was analyzed by HPLC using solvent
system I. The purity of 1 was 98.3% (retention time, 9.56 min)
with a 0.87% minor impurity identified as nortriptyline (retention
time, 8.93 min). The NMR spectrum indicated the presence of two
rotamers in an approximate 1:1 ratio. 1H NMR (300 MHz, MeOD)
ppm δ 1.81/1.90 (“quint”, J ) 7 Hz, 2H, CH2CH2CH2), 2.16-2.41
(m, 2H, CHCH2CH2), 2.45 (t, J ) 7 Hz, 2H, CH2CO2), 2.69/2.83
(s, 3H, NCH3), 2.83-3.0 (m, 4H, Ar-CH2CH2-Ar), 3.15-4.47 (m,
4H, CH2NH2, CH2NMe), 5.76-5.84 (m, 1H, CH), 6.96-7.26 (m,
8H, Ar). 13C NMR (300 MHz, MeOD) ppm δ 23.7/23.8
(CH2CH2CH2), 28.4/29.2 (CHCH2CH2), 30.8/31.5 (CH2CO), 32.9/
34.8 (Ar-CH2CH2-Ar), 33.0/34.8 (Ar-CH2CH2-Ar), 34.1/35.9
(NCH3), 40.4 (CH2NH2), 48.6/50.3 (CH2NMe), 126.8/127.0 (CH,
Ar), 127.05/127.1 (CH, Ar), 127.8/128.2 (CH, Ar), 128.4/128.7
(CH, Ar), 128.8/128.9 (CH, Ar), 129.1 (CH, Ar), 129.2/129.3 (CH,
Ar), 129.4 (CH, Ar), 131.0/131.2 (CH, Ar), 138.1 (C, Ar), 140.6/
140.9 (C, Ar), 141.8/142.2 (C, Ar), 145.9/147.2 (CdCH), 173.55/
173.7 (MeNCO). MS (ES+): m/z 349 (MH+, 100%), 332 (MH+
- NH3, 73). Anal. Calcd for C23H29ClN2O·0.5H2O: C 70.12, H
7.68, N 7.11. Found C 69.89, H 7.93, N 7.24.
Solubilization of the Drugs for the Biological Studies. Flu-
oxetine, nortriptyline, 1, 2, and gabapentin (Sigma) were solubilized
in doubly distilled water (DDW). Control animals received the same
volume of DDW, of a maximum of 0.1 mL/kg weight. The drugs
were administered to the animals by oral gavage (po) using 20-
gauge curved needles (Pop-per, NY) except gabapentin that was
given by ip injection.
Biology: Animals. Wistar male rats, 8-10 weeks old, and Balb-c
male mice, 8-12 weeks old, (Harlan, Israel) were housed under
conditions of controlled temperature (23 ( 3 °C) and humidity (55
( 15%) with a 12 h light/12 h dark cycle and were acclimated at
least 1 week prior to their use in the experiment. All animals were
fed with a commercially available rodent diet (ad libitum), and free
access to drinking water was available. All experiments were carried
out in accordance with the ethical guidelines of the Committee on
the Care and Use of Laboratory Animals of Tel Aviv University.
Hot Plate Test. The hot plate test used to measure latency in
response to heat was carried out on the basis of the method
described,20 with the following modifications. The hot plate (MRC,
model MH-4, 230 V/50 Hz, 750 W) was maintained at 52 °C.
Balb-c mice or Wistar rats were placed on the heated surface, and
the time of response to heat sensation was detected by the following
reactions of raising or licking the paw, jumping, or running and
was recorded as time (s) of response latency. Data were collected
between 0 and 5 h after po administration of the specified drugs.
Formalin Test. The method used was based on that described.22
Balb-c mice, 5-10/group, were treated orally with the tested
compounds, and 2 h later, 20 µL of a 1% formalin solution was
injected sc into the dorsal surface of their right hind paws. The
formalin induced a typical licking or biting of the injected paw
(flinching behavior). The animals were placed in a transparent
chamber, and the number of times that they licked or bit the injected
paw during the first 5 min (phase I) and between 25 and 35 min
(phase II) after the injection was counted.
N-(3-(p-(Trifluoromethyl)phenoxy)-3-phenylpropyl)-4-amino-
N-ethylbutaneamide Hydrochloride, 2. tert-Butyl 3-(N-(3-(4-
trifluoromethyl)phenoxy)-3-phenylpropyl)-N-methylcarbamoyl)pro-
pylcarbamate (N-tert-Boc-2), prepared as described in general
procedure I from fluoxetine, was obtained as a colorless oil (yield
76%) and was used as such without further purification. HPLC
analysis of the product, using solvent system II, identified purity
of 99.5% (retention time, 19.7 min). The NMR spectra of the
compound indicated the presence of two rotamers in an approximate
3:2 ratio. 1H NMR (300 MHz, CDCl3) ppm δ 1.41 (s, 9H, CMe3),
1.77 (“quint”, J ) 7 Hz, 2H, CH2CH2CH2), 2.11-2.28 (m, 4H,
CH2CO2, CHCH2CH2), 2.92/2.95 (s, 3H, NMe, minor/major), 3.05/
3.13 (t, J ) 7 Hz, 2H, CH2NH, minor/major), 3.38-3.69 (m, 2H,
CH2NMe), 5.14/5.21 (dd, J ) 8.5, 4 Hz, 1H, OCH, minor/major),
6.89 (d, J ) 8.5 Hz, 2H, Ar), 7.2-7.47 (m, 7H, Ar). 13C NMR
Measurement of TNF-r and INF-γ in the Skin of the Paws
of Mice. At the specified times shown after injection of the formalin,
the animals were killed and the skin tissue was removed from the

GABA Amides of Nortriptyline and Fluoxetine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 3017
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Lucerne, Switzerland) in 300 µL of ice-cold PBS containing 0.4
mM NaCl, 0.05% Tween-20, 0.1 mM phenylmethylsulfonyl fluoride
(PMSF), and 0.1 mM protease inhibitor cocktail (Calbiochem,
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with a permanent marker on the ankles of their left hind paws to
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(Sigma) in saline into the surface of the left hind paw. The change
in height and volume of the paw after λ-carrageenan administration
in vehicle-treated and drug-treated animals was measured using an
electronic caliper and was calculated (w2 × L/2). Latency of
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test, as described above.
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Balb-c mice were placed in the testing room 12 h prior to the
beginning of the test. The tested agents (vehicle, nortriptyline,
fluoxetine, or the GABA amides 1 or 2 at eqM doses) were
administered po 90 min prior to test. An individual mouse was
placed in the novel environment of a square open field (50 cm ×
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a camera placed above the field for 20 min and subsequently
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by Cerep, France, according to their standard operating procedures.
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of the specific ligand [3H]muscimol (Perkin-Elmer) and 10 µM of
nonradioactive 1 for 10 min at 4 °C with rat cerebral cortex. Binding
to the GABAB receptor was assayed by incubation of 2.5 nM
[3H]CGP (American Radiolabeled Chemicals, Inc.). The receptor’s
specific ligand [3H]CGP and 10 µM unlabeled 1 were incubated
for 60 min at 22 °C with HEK-293 cells, stably expressing the
human recombinant GABAB receptor. The binding to the nonselec-
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receptor’s specific ligand [3H]GABA (Perkin-Elmer) and 10 µM
unlabeled 1 for 60 min at 22 °C with rat cerebral cortex. The specific
ligand binding to the receptors was defined as the difference
between the total binding and the nonspecific binding determined
in the presence of an excess 10 µM of unlabeled 1 (in duplicate).
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Statistical Analysis. The data are expressed as the mean ( SEM
and were analyzed by the Student’s t test. Values of p < 0.05 were
considered statistically significant.
Acknowledgment. This work was partially supported by
Bioline Innovations Jerusalem LP and the “Marcus Center for
Pharmaceutical and Medicinal Chemistry” at Bar Ilan University.
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