(THIENO[2,3-b] [l,5]BENZOXAZEPIN-4-YL)PIPERAZIN-l-YL COMPOUNDS AS DUAL ACTIVITY HI INVERSE AGONISTS/5-HT2A ANTAGONISTS
Histamine plays an important role in a variety of physiological processes through
its interaction with at least four different G-protein coupled receptors, the H1-H4
receptors. In the CNS, HI receptors play a key role in the sleep regulation cycle and HI
antagonists/inverse agonists are known to induce somnolence.
Likewise, serotonin plays important roles in a variety of physiological processes
through its interaction with at least fourteen different G-protein coupled receptors.
Modulation of 5-HT2Areceptors in the CNS plays a key role in the sleep regulation cycle
and 5-HT2Aantagonists have been shown to improve slow wave sleep and sleep
maintenance in patients with insomnia.
Compounds having HI or 5-HT2Ainverse agonist or antagonist activity have been
used in the treatment of insomnia (e.g. doxepin and trazodone, respectively) and have
exhibited significant pharmacological effects in animal sleep studies. However, no
selective dual activity H 1/5-HT2Ainverse agonists/antagonists are currently commercially
available.
WO 2007/022068 describes certain substituted (thieno[2,3-b][l,5]benzodiazepine-
4-yl)piperazin-l-yl and (thieno[2,3-b][l,5]benzoxazepine-4-yl)piperazin-l-yl compounds
for treating sleep disorders.
The present invention provides 3-[4-(2-chloro-8-methyl-thieno[2,3-
b][l,5]benzoxazepin-4-yl)piperazin-l-yl]-2,2-dimethyl-propanoic acid and
pharmaceutically acceptable salts thereof, having high inverse agonist potency for the HI
receptor, high antagonist potency for the 5-HT2A receptor, and good selectivity for these
receptors, particularly as against other histamine receptors, serotonin receptors and other
physiologically relevant receptors, particularly as against the 5-HT2C receptor, GABAA
receptor, muscarinic receptors, dopaminergic receptors, adrenergic receptors, and the
hERG channel. These compounds also demonstrate through animal models that they may
be useful for the treatment of sleep disorders characterized by poor sleep maintenance.
As such, the compounds are believed to be useful for the treatment of sleep disorders
characterized by poor sleep latency or poor sleep maintenance or both, such as the
treatment of insomnia, as for example chronic or transient primary insomnia, or chronic
or transient secondary insomnia, or both. Examples of secondary insomnia include, but
are not limited to insomnia associated with depressive disorders (e.g. major depressive
disorder, dysthymia, and/or cyclothymia), insomnia associated with anxiety disorders
(e.g. generalized anxiety disorder and/or social phobia), insomnia associated with pain
(e.g. fibromyalgia, chronic bone or joint pain, such as associated with inflammatory
arthritis or osteoarthritis, or diabetic neuropathic pain), insomnia associated with allergic
reactions (e.g. allergic asthma, pruritus, rhinitus, congestion, etc.), insomnia associated
lung or airway disorders (e.g. with obstructive sleep apnea, reactive airway disease, etc.),
insomnia associated with psychiatric disorders, dementia, and/or neurodegenerative
diseases, and/or insomnia associated with circadian rhythm sleep disorders (e.g. shift
work sleep disorder, jet lag disorder, delayed sleep phase disorder, advanced phase sleep
disorder, and non-24 hr. sleep-wake syndrome, etc.).
Further, the compounds of the present invention demonstrate potentiation of their
effects on non-rapid eye movement sleep ( REM sleep) when coadministered with
selective serotonin reuptake inhibitors.
The present invention provides a compound of Formula I
I
or a pharmaceutically acceptable salt thereof. That is to say 3-[4-(2-chloro-8-
methylthieno[2,3-b][l,5]benzoxazepin-4-yl)piperazin-l-yl]-2,2-dimethylpropanoic acid
or a pharmaceutically acceptable salt thereof.
In another aspect of the invention there is provided a pharmaceutical composition
comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, in
combination with at least one pharmaceutically acceptable carrier, diluent, or excipient.
Furthermore, this aspect of the invention provides a pharmaceutical composition for
treating insomnia, as for example insomnia characterized by prolonged sleep latency or
poor sleep maintenance or both, as for example primary insomnia, jet lag, shift work
sleep disorder, delayed sleep phase disorder, advanced phase sleep disorder, and/or non-
24 hr. sleep-wake disorders, comprising a compound of Formula I or a pharmaceutically
acceptable salt thereof, in combination with one or more pharmaceutically acceptable
excipients, carriers, or diluents.
A further embodiment of this aspect of the invention provides a pharmaceutical
composition comprising a compound according to Formula I, or pharmaceutically
acceptable salt thereof, in combination with at least one pharmaceutically acceptable
carrier, exciepient or diluent, and optionally other therapeutic ingredients. In a yet further
embodiment of this aspect of the invention, the pharmaceutical composition further
comprises a second therapeutic agent which is a serotonin reuptake inhibitor, as for
example citalopram, paroxetine, fluoxetine and/or fluvoxetine.
The present invention also provides a method of treating insomnia, as for example
insomnia characterized by prolonged sleep latency or poor sleep maintenance or both, as
for example primary insomnia, jet lag, shift work sleep disorder, delayed sleep phase
disorder, advanced phase sleep disorder, and/or non-24 hr. sleep-wake disorders, in a
mammal comprising administering to a mammal in need of such treatment an effective
amount of a compound of Formula I or a pharmaceutically acceptable salt thereof. In
another embodiment of this aspect of the invention, the method further comprises
administering in simultaneous, separate or sequential combination, a second therapeutic
agent which is a serotonin reuptake inhibitor, as for example citalopram, paroxetine,
fluoxetine and/or fluvoxetine. In one particular embodiment of these methods of
treatment, the mammal is a human.
This invention also provides a compound of Formula I or a pharmaceutically
acceptable salt thereof for use in therapy. Within this aspect, the invention provides a
compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in the
treatment of insomnia. In further embodiments, the insomnia is characterized by
prolonged sleep latency or poor sleep maintenance or both, as for example primary
insomnia, jet lag, shift work sleep disorder, delayed sleep phase disorder, advanced phase
sleep disorder, and/or non-24 hr. sleep-wake disorders. In another embodiment of this
aspect, the invention provides a compound according to Formula I, or a pharmaceutically
acceptable salt thereof, for use in simultaneous, separate or sequential combination with a
serotonin reuptake inhibitor, as for example citalopram, paroxetine, fluoxetine and/or
fluvoxetine, in the treatment of insomnia. One particular embodiment of this aspect of
the inventions, the uses are in mammals, particular humans.
Another aspect of this invention provides the use of a compound of Formula I, or
a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the
treatment of insomnia, as for example primary insomnia characterized by prolonged sleep
latency or poor sleep maintenance or both, as for example primary insomnia, jet lag, shift
work sleep disorder, delayed sleep phase disorder, advanced phase sleep disorder, and/or
non-24 hr. sleep-wake disorders. Another embodiment of this aspect of the invention
provides the use of a compound of Formula I, or a pharmaceutically acceptable salt
thereof, and a second therapeutic agent which is a serotonin reuptake inhibitor, as for
example citalopram, paroxetine, fluoxetine and/or fluvoxetine, in the manufacture of a
medicament for the treatment of insomnia, as for example, insomnia characterized by
prolonged sleep latency and/or poor sleep maintenance, as for example primary insomnia,
jet lag, shift work sleep disorder, delayed sleep phase disorder, advanced phase sleep
disorder, and/or non-24 hr. sleep-wake disorders.
For clarity, the following numbering of the tricyclic ring structure will be used
throughout the application:
The compound of this invention has basic and acidic moieties, and accordingly
reacts with a number of organic and inorganic acids and bases to form pharmaceutically
acceptable salts. Pharmaceutically acceptable salts of the compound of the present
invention are contemplated within the scope of the present application. The term
"pharmaceutically acceptable salt" as used herein, refers to any salt of a compound of the
invention that is substantially non-toxic to living organisms. Such salts include those
listed in Journal of Pharmaceutical Science, 66, 2-19 (1977), which are known to the
skilled artisan.
Abbreviations used herein are defined as follows:
"DMEM" means Dulbecco's Minimum Eagle's Medium.
"DMSO" means dimethyl sulfoxide.
"EDTA" means ethylenediaminetetraacetic acid.
"FBS" means fetal bovine serum.
"HEPES" means 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid.
"HPLC" means high pressure liquid chromatography.
"hr." means hours.
"IC50" means the concentration at which 50% of the maximum inhibition is
achieved.
"LC-MS" means HPLC-mass spectrography.
"MeOH" means methanol.
"min." means minutes.
"MS" means mass spectroscopy.
"MS (ES+)" means mass spectroscopy using electrospray ionization.
"NMR" means nuclear magnetic resonance.
"THF" means tetrahydrofuran.
General Chemistry
The compound of the present invention can be prepared according to the
following synthetic examples.
Preparation 1. 2,5-dichlorothiophene-3-carbonyl chloride
To a suspension of 2,5-dichloro-thiophene-3-carboxylic acid (49.7 g; 252.23
mmoles; 1.00 equiv) in dichloromethane (500 mL), add dimethylformamide (0.5 mL;
6.47 mmoles) followed by a solution of 2 M oxalyl chloride in dichloromethane (138.73
mL; 277.45 mmoles; 1.1 equiv) over 1.5 hr. (vent the evolved gas through a caustic
solution). Stir the resulting clear solution at room temperature for 1 hr. until gas
evolution has ceased and the reaction is complete by LCMS (quench a sample into 7 M
NH3/MeOH for reaction monitoring) MS (m/z): = 195.9, 197.9 (M+H)+ for
corresponding primary amide. Evaporate to dryness to give the title intermediate as a
brown oil (55g, 252 mmol, quantitative).
Preparation 2. 2,5-dichloro-N-(2-hydroxy-4-methyl-phenyl)thiophene-3-
carboxamide
To a solution of 6-amino-m-cresol (34.14 g; 277.20 mmoles; 1.1 equiv) in THF
(450 mL), add pyridine (40.76 mL; 504.00 mmoles; 2 equiv), followed by a solution of
2,5-dichlorothiophene-3-carbonyl chloride (54.30 g, 252 mmoles, 1.00 equiv) in THF
(250 mL) over 30 min., using an ice-bath to maintain a temperature of 15-20°C. Stir the
resulting thick mixture at room temperature for 1 hr. to give complete consumption of the
aminophenol by LC-MS. Pour onto a mixture of 2 M aqueous HC1 (500 ml) and ice (250
ml) with agitation. Collect the resulting beige solid by filtration, wash well with water,
and dry in air. MS (m/z): = 301.84, 303.94 (M+H)+. Dry in a vacuum oven at 40°C over
P2O 5 overnight to give the title intermediate (84. 5g, assumed quantitative).
Preparation 3. 2-chloro-8-methyl-5H-thieno[2,3-b] [l,5]benzoxazepin-4-one
To a well-stirred suspension of 2,5-dichloro-N-(2-hydroxy-4-methylphenyl)
thiophene-3-carboxamide (76.15 g; 252 mmoles; 1.00 equiv) in dimethyl
sulfoxide (450 mL), add potassium carbonate (38.31 g; 277.20 mmoles; 1.1 equiv) and
heat the mixture to 100-1 10°C for 4.5 hr. to give essentially complete conversion by
LCMS. Allow to cool to room temperature and slowly add to two separate beakers
containing 1M aqueous hydrochloric acid (500 ml), observing gas evolution. Stir at
room temperature for 0.5 hr. and collect the resulting dark grey solid by filtration. Wash
sequentially with water, followed by a small amount of ethanol, followed by a small
amount of diethyl ether. Dry in a vacuum oven at 45°C overnight to give the title
intermediate (58.5g, 87%). MS (m/z): = 265.99 (M+H)+.
Preparation 4. 2,4-dichloro-8-methyl-thieno[2,3-b] [l,5]benzoxazepine
Charge a 1 L round bottom flask with methoxybenzene (225 mL, 5V), 2-chloro-8-
methyl-5H-thieno[2,3-b][l,5]benzoxazepin-4-one (45 g; 169.4 mmoles; 1 equiv) and
N,N-dimethylaniline (47.2 g; 389.5 mmoles; 2.3 equiv). Heat to 60°C and add
phosphoryl chloride (85.7 g; 558.9 mmols; 3.3 equiv) drop wise over 0.5 hr. Warm up to
100°C and stir for 2 hr. until complete by TLC analysis. Cool to 40-60°C and evaporate
to obtain the title intermediate as a dark brown solid (123.1 g, 433.2mmoles, 256% yield
uncorrected by assay). MS (m/z): 283.8 (M+H).
Preparation 5. Methyl 3-[4-(2-chloro-8-methyl-thieno[2,3-b][l,5]benzoxazepin-4-
yl)piperazin-l-yl]-2,2-dimethyl-propanoate
Charge a 1 L round bottom flask with 2,4-dichloro-8-methyl-thieno[2,3-
b][l,5]benzoxazepine (121.1 g; 169.4 mmols; 1.0 equiv), followed by acetonitrile (600
mL, 12.5V), then potassium carbonate ( 119.4 g; 863.9 mmols) in one portion. Stir for 10-
20 min. and then add methyl 2,2-dimethyl-3-(piperazin-l-yl)propanoate dihydrochloride
(55.54 g; 203.3 mmoles; 1.2 equiv) in one portion. Heat to 80°C and stir for 30 hr.
Concentrate the mixture to dryness under vacuum, then charge ethyl acetate (1920 mL,
40V) and water (1920 mL, 40V) into the mixture. Stir, filter and then separate the water
phase and extract with ethyl acetate (960 mL, 20V). Combine the organic phases and
wash with water (960 mL 2) and brine (200 mL, 4V). Concentrate and purify by silica
gel column (petroleum ether/ethyl acetate (0 to 10%)) to obtain the title intermediate as
yellow solid (55.4 g, 123.7 mmoles, 95.8% purity, 73.0% yield uncorrected by assay).
MS (m/z): 448.2 (M+H). ¾ NMR (400MHz,CDCl 3) : 57.03 (m, IH), 6.91 (m, IH), 6.85
(s, IH), 6.50 (s, IH) , 3.67(s, 3H), 3.47 (m, 4H), 2.58-2.54 (m, 6H), 2.28 (s, 3H), 1.19 (s,
6H).
Example 1. 3-[4-(2-Chloro-8-methyl-thieno[2,3-b] [l,5]benzoxazepin-4-yl)piperazinl-
yl]-2,2-dimethyl-propanoic acid
Charge a 2 L round bottom flask with methyl 3-[4-(2-chloro-8-methyl-thieno[2,3-
b][l,5]benzoxazepin-4-yl)piperazin-l-yl]-2,2-dimethyl-propanoate (70.3 g; 156.9
mmoles; 1.0 equiv), isopropyl alcohol (469 mL; 6.67V) and water (469 mL; 6.67V).
Then add sodium hydroxide (18.83g; 470.8 mmoles; 3.0 equiv) and heat the mixture to
80°C with stirring for 3 hr. Cool to 20°C and neutralize to pH 7 with 5 M aqueous HCl.
Evaporate most of the isopropyl alcohol then adjust the pH to 6-7 with 5 M aqueous HCl
and concentrate to remove solvents. Charge ethyl acetate (2800 mL, 40V) and water
(2800 mL, 40V) then stir for 30 min. before filtering to obtain crude product. Separate
the water phase and extract with ethyl acetate (700 mL, 10V). Combine ethyl acetate
layers and evaporate to obtain crude product. Charge the crude product and isopropyl
alcohol (500 mL, 7V) into 1 L round bottom flask. Heat to 80°C and stir for 1 hr., then
cool to 20°C slowly. Filter and wash the cake with isopropyl alcohol (70 mL, 1V) to
obtain the title compound as yellow solid (60.4 g; 138.6 mmoles; 99.3% purity, 88.3%
yield corrected by assay). MS (m/z): 434.0 (M+H). H NMR (400MHz, CDC13) : 57.01
(m, 1H), 6.94 (m, 1H), 6.82 (s, 1H), 6.50 (s, 1H), 3.64 (brs, 4H), 2.86 (brs, 4H), 2.60 (s,
2H), 2.29 (s, 3H), 1.26 (s, 6H). 13C NMR (400MHz, CDC13) : 5178.57, 161.10, 154.37,
152.00, 136.63, 135.64, 127.95, 127.14, 121.88, 121.20, 120.14, 118.58, 65.70, 54.46,
46.45, 41.54, 25.34, 20.70.
Example 2. 3-[4-(2-chloro-8-methyl-thieno[2,3-b] [l,5]benzoxazepin-4-yl)piperazinl-
yl]-2,2-dimethyl-propanoic acid
To a suspension of methyl 3-[4-(2-chloro-8-methyl-thieno[2,3-
b][l,5]benzoxazepin-4-yl)piperazin-l-yl]-2,2-dimethyl-propanoate (22.5 g; 50.23
mmoles; 1.00 equiv) in a mixture of isopropyl alcohol (150 mL; 1.96 moles) and water
(150 mL) add sodium hydroxide (6.03 g; 150.68 mmoles; 3 equiv) and heat the mixture at
80°C (oilbath) for 2.5 hr. to give complete conversion by LC-MS. Allow to cool and
neutralize to pH 7 with 5 M aqueous HCl. Evaporate most of the isopropyl alcohol to
give a fine precipitate. Readjust the pH with 5 M aqueous HCl to pH 7. Stand the flask
in a refrigerator for 0.5 hr. then collect the pale-yellow solid by filtration, washing with
water. Dry in a vacuum oven at 45°C over P 2O overnight to give the title compound
(20.6g, 95%). MS (m/z): = 434. 1 (M+H)+.
Example 3. 3- [4-(2-chloro-8-methyl-thieno[2,3-b][l,5]benzoxazepin -4-yl)piperazinl-
yl]-2,2-dimethyl-propanoic acid dihydrochloride
Slurry 3-[4-(2-chloro-8-methyl-thieno[2,3-b][l,5]benzoxazepin-4-yl)piperazin-lyl]-
2,2-dimethyl-propanoic acid (5.8 g; 13.3 mmol) in a small amount of acetonitrile,
then add 4 M dioxane solution of hydrogen chloride (14.06 g; 53.57 mmoles; 4 equiv)
and evaporate the resulting solution to dryness. Triturate with a small amount of diethyl
ether, collect by filtration and dry in a vacuum oven to give the title compound (4.74g,
70%); MS (m/z): = 434.1 (M+H)+.
Additional purification of the dihydrochloride salt
Take 3-[4-(2-chloro-8-methyl-thieno[2,3-b][l,5]benzoxazepin-4-yl)piperazin-lyl]-
2,2-dimethyl-propanoic acid dihydrochloride (7.5 g; 14.80 mmoles; 1.00 equiv) and
heat in ethanol (150 ml) with sonication until a complete mixture is obtained. Evaporate
to dryness. Triturate with a small amount of diethyl ether and collect the beige solid by
filtration. Grind to a fine powder and dry in a vacuum oven at 50°C over 2 nights to give
the title compound (7.14g, 95%). MS (m/z): = 434.1 (M+H)+.
Literature data (Morairty SR, Hedley L, Flores J, Martin R, Kilduff TS. (2008)
Selective 5-HT2A and 5-HTe receptor antagonists promote sleep in rats. Sleep 31, 34-44.;
and Barbier, A.J., and Bradbury, M.J., Histaminergic Control of Sleep-Wake Cycles:
Recent Therapeutic Advances for Sleep and Wake Disorders, CNS & Neurological
Disorders - Drug Targets, vol 6, pg. 3 1-43 (2007)) and data generated in non-clinical
animal studies support a role for dual activity HI inverse agonists / 5-HT2A antagonists in
the treatment of insomnia and in the symptomatic treatment of insomnia associated with
other disorders such as depressive disorders, anxiety disorders, pain, allergies, lung or
airway disorders, psychiatric disorders, dementia, and/or neurodegenerative diseases,
and/or circadian rhythm sleep disorders. Specifically it is found that certain dual activity
HI inverse agonists / 5-HT2A antagonists are effective in increasing total sleep time using
EEG monitored rodents without disproportionate or clinically relevant hypoactivity,
decrease in REM sleep, or hypersomnolence.
To further demonstrate the characteristics of the present compounds, they may be
run in the following in vitro and in vivo assays:
In vitro binding and activity assays:
HI competition binding assay
[ H]-Pyrilamine binding experiments are carried out in SPA (scintillation
proximity assay) 96-well format. Membranes used in this assay are prepared from HEK-
293 cells stably expressing recombinant HI receptor (human). The incubation is initiated
by the addition of a mixture of WGA PVT SPA beads (lmg/well, Perkin Elmer (MA,
USA) RPNQ0001) and 3 mg membranes to assay buffer (67 mM Tris; pH 7.6) containing
3.5 nM [ H]-Pyrilamine and varying concentrations of the test compound (10 point
concentration response curves). Non-specific binding is determined in the presence of 10
mM Triprolidine. Samples are incubated for 4 hr. at room temperature (22° C) and then
read in a Microbeta Trilux.
5-HT competition binding assay
[ H]-Ketanserin binding experiments are carried out in SPA 96-well format.
Membranes used in this assay are prepared from AV-12 cells stably expressing
recombinant 5-HT2A receptor (human). The incubation is initiated by the addition of a
mixture of WGA YSi SPA beads (lmg/well, Perkin Elmer (MA, USA), RPNQ001 1) and
2 mg membranes to assay buffer (67 mM Tris, 0.5 mM EDTA; pH 7.6) containing 3.1 nM
[ H]-Ketanserin and varying concentrations of the test compound (10 point concentration
response curves). Non-specific binding is determined in the presence of 20 mM 1-(1-
Naphthyl) piperazine. Samples are incubated for 4 hr. at room temperature (22° C) and
then read in a Microbeta Trilux.
-HT competition binding assay
[1 I]-(±)DOI binding experiments are carried out in SPA 96-well format.
Membranes used in this assay are prepared from AV-12 cells stably expressing
recombinant 5-HT2C receptor (human). The incubation is initiated by the addition of a
mixture of WGA PVT SPA beads (0.5 mg/well, Perkin Elmer (MA, USA), RPNQ0001)
and 2.5 mg membranes to assay buffer (50 mM Tris-HCl, 10 mM MgCl 2, 0.5 mM EDTA,
10 mM pargyline, 0.1% ascorbic acid, pH7.4) containing 0.2 nM [[1 I]-(±)DOI and
varying concentrations of the test compound (10 point concentration response curves).
Non-specific binding is determined in the presence of 20 mM l-(l-Naphthyl) piperazine.
Samples are incubated for 4 hr. at room temperature (22° C) and then read in a Microbeta
Trilux.
Binding data analysis
Curves are evaluated using a 4-parameter logistic nonlinear equation to obtain the
concentration of competitor causing 50% inhibition of radioligand binding (IC50) .
Equilibrium dissociation constants (¾) are calculated according to the equation K =
IC5o/(l+L/K d), where L equals the concentration of radioligand used in the experiment
and K equals the equilibrium dissociation constant of the radioligand for the receptor,
determined from standard saturation analysis or homologous competition experiments.
Reported values for ¾ , where n values are indicated, are shown as geometric mean ± the
standard error of the mean (SEM), with the number of replicate determinations indicated
by n. Geometric means are calculated by the equation GeoMean = 10 (Average (log ¾ 1
+ log ¾ 2 + . . .log ¾ n)/sqrt n).
GABAA antagonism using native receptors in primary neuronal cultures
Activity of compounds on native GABAA receptors is evaluated by monitoring
calcium fluxes using a 96 well format FLIPR® system (Fluorometric Imaging Plate
Reader (FLIPR®, Molecular Devices). Briefly, cortical embryonic neurons are
dissociated from El 8 rat embryos and plated at optimum density into black-walled,
transparent bottom poly-D-lysine coated 96-well FLIPR® plates. After loading the cells
with a calcium sensitive dye (Fluo4-AM, Molecular Devices), the cells are bathed in a
solution containing low chloride (chloride replaced by gluconate). Under these
conditions activation of GABAA receptors causes an efflux of chloride ions (in the
direction of the chemical gradient), which results in membrane depolarization and
consequently activation of voltage gated calcium channels (VGCCs). Calcium influx
through VGCCs is recorded and analysed offline using the FLIPR® system. For a
pharmacological validation of the assay, concentration response curves (CRC) are
recorded for the standard agonist (GABA) and standard antagonist (Gabazine). Any
effects are determined in CRC mode against a fixed concentration of agonist GABA at 10
mM (equivalent to an EC90 GABA response).
Methods:
The antagonist effects of compounds are quantified using 10-point dose response
curves by comparing the peak fluorescent responses to the agonist GABA in the presence
and absence of compound. The assay window is defined as the maximal response
obtained by GABA at its predetermined EC90 concentration minus the response obtained
by a fully inhibiting concentration of gabazine (50 mM) . Antagonist effects are calculated
as a percent of the assay window. All data are calculated as relative IC50 values using a
four-parameter logistic curve fitting program (Prism Graphpad® 3.01). Antagonist
potencies for all compounds are compared to gabazine with three replicates in each assay
run.
Further, the compounds of the invention may be tested in binding assays and
functional activity assays by well known methods for other physiologically important
receptors such as, but not limited to, the hERG channel, other serotonin receptors
(specifically 5-HTi B, 5-HTiD, receptors, lack of agonist activity at 5-HT2Breceptors, 5-
HT2C, 5-HT5, 5-HT6, and 5-HT7 receptors), dopaminergic receptors (specifically Dl, D2,
and D3), GABAA receptors, adrenergic receptors and monoamine transporters.
The compounds of examples 1 and 13 are tested essentially as described above
and are found to have activity profiles as shown in Table 1.
Table 1. Selectivity data
Example 1 Example 3
HI ¾ (nM) 20.6 58.8
5-HT2A Ki (nM) 3.37 7.01
5-HT2B Ki (nM) 121
5-HT2B Agonist EC 0 (nM) - >10,000
5-HT2B Antagonist Kb (nM) 78.6
5-HT2CKi (nM) 137 328
GABAA ICJO (mM) >100
hERG Channel (mM) >100
Dopamine Di ¾ (nM) 592
Dopamine D2 ¾ (nM) 2780 >4570
Dopamine D ¾ (nM) >5510 >5680
5-HTIB (nM) >5580
5-HT1D Ki (nM) >3980
5-HT5 ¾ (nM) >8810
5-HT6Ki (nM) >5830
5-HT7Ki (nM) >2060
Adrenergic alphas i (nM) >10200
Adrenergic alphas ¾ (nM) >14700
Adrenergic alpha2A i (nM) >8990
Adrenergic alpha2B ¾ (nM) >5760
Adrenergic alpha2c ¾ (nM) >4230
Serotonin Transporter >661
Norepinephrine Transport >696
Dopamine Transporter >871
Therefore, physiologically relevant doses of the compounds of the invention are
expected to provide substantial inhibition of HI and 5-HT2A receptors in vivo, while not
substantially interacting with other physiologically relevant receptors, and thus are
expected to provide the desired pharmacology while avoiding undesired effects associated
with off-target activity. Such undesired effects include, but are not limited to the
following: 5-HT2 C antagonist activity associated with treatment emergent weight gain, 5-
HT2B agonist activity associated with valvulopathy, hERG channel modulation associated
with QT prolongation, and GABAA antagonist activity associated with seizure activity.
Furthermore, interference with sleep/wake physiology is avoided by the selectivity over
dopamine receptors, other serotonin receptors, adrenergic receptors, and monoamine
transporters.
5-HTTA Receptor Occupancy: Receptor occupancy is assayed to demonstrate
antagonist/inverse agonist activity at the 5-HT2AReceptor in vivo. Briefly, male Sprague-
Dawley rats (Harlan Sprague-Dawley, Indianapolis, GN ) weighing approximately 230-280
grams are given ad lib access to food and water until the beginning of the 3-hr.
experimental protocol. 1 mg/kg ketanserin (non-selective 5-HT2A antagonist) is used as a
positive control to establish assay validity. Test compounds or control are administered
by oral gavage in a vehicle comprised of 20% hydroxypropyl beta-cyclodextrin. MDL
100907 ( (R)-(+)-a-(2,3-Dimethoxyphenyl)-l-[2-(4-fluorophenyl)ethyl]-4-
piperidinemethanol), a selective 5-HT2A antagonist, is used as a tracer. MDL 100907 is
suspended in water with 5 mΐ dilute lactic acid ( 1 mg/ml), diluted to 6 mg/ml with saline,
and administered in a volume of 1 mL/kg intravenously via the lateral tail vein to yield a
tracer dose of 3 mg kg. Rats are administered test compound, ketanserin, or vehicle (N =
4), followed 1 hr. later with an intravenous, 3 mg/kg tracer dose of MDL 100907. It is at
the time of tracer administration that receptor occupancy (RO) is considered to be
measured. Fifteen min. after tracer administration, rats are sacrificed by cervical
dislocation. Plasma samples are collected and samples of the frontal cortex and
cerebellum are removed. The level of MDL 100907 tracer is measured in each cortical
and cerebellar sample. RO is calculated using the well-established ratio method which
employs a region of high receptor density representative of total binding (frontal cortex)
normalized by an area without or with very low levels of receptor (cerebellum). This
region, referred to as the null region, represents nonspecific binding of the ligand probe.
Vehicle ratio of the tracer levels in cortex relative to cerebellum represents 0%
occupancy. A ratio of 1 represents 100% occupancy and is achieved when all specific
binding to the 5-HT2A receptor of the MDL 100907 tracer is blocked. The intermediate
ratios of cortical to cerebellar tracer from the test compound pretreated group are
interpolated linearly between the ratio of tracer levels in the vehicle-treated animals (0%
occupancy) and a ratio of 1 (100% occupancy) in order to determine the percent 5-HT2A
RO.
MDL 100907 Analysis: Cortex and cerebellar samples are weighed and placed in conical
centrifuge tubes on ice. Four volumes (w/v) of acetonitrile containing 0.1% formic acid
is added to each tube. The samples are then homogenized and centrifuged at 14,000 RPM
(21,920 x g) for 16 min. Supernatant is diluted by adding 100 - 900 m sterile water in
HPLC injection vials for LC/MS/MS analysis. Analysis of MDL 100907 is carried out
using an Agilent model 1200 HPLC (Agilent Technologies, Palo Alto, CA) and an API
4000 mass spectrometer. The chromatographic separation is on a 2.1 X 50 mm C18
column (Agilent part number 971700-907) with a mobile phase consisting of 60%
acetonitrile in water with an overall 0.1% formic acid content. Detection of MDL 100907
is accomplished by monitoring the precursor to product ion transition with a mass to
charge ratio (m/z) of 374.2 to 123.0. Standards are prepared by adding known quantities
of analyte to brain tissue samples from non-treated rats and processing as described
above.
Statistical Methods : Curves for each study are fitted to a 4 parameter logistic function
with the bottom fixed at 0% using JMP® version 8.0 (SAS Institute Inc, Cary NC) and
the absolute ED50 is calculated by the software. Values are given as means, standard
errors and 95% confidence intervals. The compound of Example 3 is tested essentially as
described and is found to achieve high 5-HT2Areceptor occupancy with an ED 0 of 0.09
mg/kg.
HI Inverse Agonism: To determine the inverse agonist nature of compounds of the
present invention, their effects on the levels of Myo-Inositol 1 phosphate (IP1) in
HEK293 cells transfected with the human recombinant HI receptor (HEK293/hm HI
clone R-40) are measured. Briefly, HEK293/hm HI cells (clone R-40) are grown to
-90% confluency (3:1 DMEM/F12, 5% FBS, 20 mM HEPES, G418 500 m iΐ , 1%
Pen/Strep/Glutamine) and harvested on the day of the assay using l x Trypsin/EDTA
(PAA Pasching, Austria L I 1-003). 35 mΐ cells (300K) are seeded into 96W half area
white solid bottom plates (Corning, UK 3688) in stimulation buffer (NaCl 146 mM,
CaCl2 1 mM, KC1 4.2 mM, MgCl2 0.5 mM, Glucose 5.5 mM, HEPES 10 mM and LiCl
50 mM). Test compounds are initially dissolved in 100% DMSO at lOOx final
concentration. These are further diluted to x2 final assay concentration in stimulation
buffer and 35 mΐ of this solution is then added to the cells in the assay plate. Cells plus
compound are incubated for 1 hr. 30 min. at 37 C / 5% C0 2 before addition of 15 mΐ of
each of the HTRF IP1 detection kit reagents (CisBio 62P1APEC). The cell plate is
incubated for a further hour at room temperature before measuring IP 1 accumulation
(Envision plate reader, Perkin Elmer). IP1 accumulation (nM) is calculated by
extrapolation from the standard IP1 curve run on the day of the assay. Negative efficacy
values are expressed relative to the positive control Tripelennamine (10 mM, Sigma, UK
P5514). Compound 3 is tested essentially as described and is found to fully suppress
constitutive activity (105% at 1 mM and 85% at 10 mM (n=2)).
Inhibition of DOI Induced Headshake Activity: The in vivo 5-HT2A receptor antagonist
activity of the compound of the present invention is further demonstrated by its ability to
block head shaking activity induced by the 5-HT2A receptor agonist 2,5-dimethoxy-4-
iodoamphetamine (DOI). (see for example Bartoszyk GD, van Amsterdam C, B5ttcher
H, Seyfried CA. EMD 281014, a new selective serotonin 5-HT2A receptor antagonist.
Eur J Pharmacol. 2003 473: 229-230.) Briefly, male C57BL/6J mice (20-25 g, Charles
River) are housed in standard housing conditions (32 mice in a large IVC cage, 07.00 to
19.00 light phase, constant temperature (19-23°C) and humidity (50% +/-10), ad lib food
and water). Mice received either vehicle (0.25% Methyl cellulose), DOI (3 mg/kg in
saline) or test compound at lOmg/kg PO plus DOI (3 mg/kg in saline). Test compounds
are individually evaluated in groups of four per experiment with n=4 for each compound,
together with vehicle and DOI+vehicle (n=8). After a test compound pre-treatment time
of 60 min. the mice receive either vehicle (saline) or 3 mg/kg DOI dosed subcutaneous ly,
and are then placed into clear perspex observation chambers. Five min. after DOI or
vehicle administration the number of visually scored head shakes exhibited by each
individual mouse is counted for 15 min. The data is analyzed using an ANOVA and posthoc
Dunnet's Test. The compound of example 3 is tested essentially as described and is
found to inhibit the DOI induced headshake response at 100% at 10 mg/kg.
Sleep and behavioral monitoring in rats: The compound of the present invention is tested
in rats for its ability to increase the amount of sleep or decrease sleep interruption or both
without undesired effects such as inhibition of REM sleep, waking motor impairment,
and/or rebound insomnia. Test animals are continuously monitored by electro
encephalograms (EEG), electromyograms (EMG), and motion to measure cumulative
nonREM sleep, cumulative total sleep, average sleep bout duration, longest sleep bout
duration, rebound insomnia, REM sleep inhibition and locomotor activity intensity during
wakefulness. Methods for such studies are known in the art (see for example methods
described in Edgar DM, Seidel WF. Modafinil induces wakefulness without intensifying
motor activity or subsequent rebound hypersomnolence in the rat. J Pharmacology &
Experimental Therapeutics 1997; 283: 757-769; van Gelder R , Edgar DM, Dement
WC. Real-time automated sleep scoring: validation of a microcomputer-based system
for mice. Sleep 1991, 14: 48-55; and Gross BA, Walsh CM, Turakhia AA, Booth V,
Mashour GA, Poe GR. Open-source logic-based automated sleep scoring software using
electrophysiological recordings in rats. J Neurosci Methods. 2009; 184(1): 10-8.)
Studies are conducted as follows:
Animal preparation. Adult, male Wistar rats (approximately 270-300 g at time of
surgery) are surgically fitted for chronic recording of EEG, EMG, and motion as follows:
Rats are surgically prepared with a cranial implant consisting of four stainless steel
screws for EEG recording (two frontal [3.9 mm anterior from bregma, and ±2.0 mm
mediolaterally] and two occipital [6.4 mm posterior from bregma, ±5.5 mm
mediolaterally]), and with two Teflon-coated stainless steel wires for EMG recording
(positioned under the nuchal trapezoid muscles). All leads are soldered to a miniature
connector (Microtech, Boothwyn, PA) prior to surgery. The implant assembly is affixed
to the skull by the combination of the stainless steel EEG recording screws, cyanoacrylate
applied between the implant connector and skull, and dental acrylic. Locomotor activity
is monitored via a miniature transmitter (Minimitter PDT4000G, Philips Respironics,
Bend, OR) surgically placed into the abdomen. At least 3 weeks are allowed for
recovery.
Recording environment. Each rat is housed individually within a microisolator cage
modified with an inserted polycarbonate filter-top riser to allow more vertical headroom.
A flexible cable that minimally restricts movement is connected at one end to a
commutator afixed to the cage top and at the other end to the animal's cranial implant.
Each cage is located within separate, ventilated compartments of a stainless steel sleepwake
recording chamber. Food and water are available ad libitum and the ambient
temperature is maintained at about 23±1°C. A 24-hr light-dark cycle (LD 12:12) using
fluorescent light is maintained throughout the study. Relative humidity averages
approximately 50%. Animals are undisturbed for at least 30 hrs before and after each
treatment.
Study design and dosing. The vehicle (placebo, methylcellulose 15 centipoise 0.25% in
water) or one of the test compound dose levels is administered orally at 1 mL/kg pseudorandomly
such that no rat receives the same treatment twice, and no rat receives more
than two of the 8 treatments in any one study. Each rat is removed from its cage for about
a minute to be weighed and treated. At least 6 days "washout" period precede and follow
each treatment.
Data collection. Sleep and wakefulness discrimination may be automated (e.g., Van
Gelder et al. 1991 (above); Edgar et al. 1997 (above); Winrow CJ, et al,
Neuropharmacology 2010; 58(1): 185-94.; and Gross et al, 2009 (above). EEG is
amplified and filtered (X10,000, bandpass 1-30 Hz), EMG is amplified and integrated
(bandpass 10-100 Hz, RMS integration), and non-specific locomotor activity (LMA) is
monitored simultaneously. Arousal states are classified in 10 second epochs as non-REM
sleep, REM sleep, wakefulness, or theta-dominated wakefulness. Locomotor activity
(LMA) is recorded as counts per minute and is detected by commerically available
telemetry receivers (ER4000, Minimitter, Bend, OR).
Statistical Analysis. All animals having at least one outcome are included in the summary
results (for example, we include appropriate data from an animal treatment for which
telemetry data is usable but EEG data is not). The post-treatment observation period is
divided into post-dosing intervals appropriate to each Outcome, where the time of dosing
is defined as the start of Hour = 0, and outcomes are summarized in the observation
period by computing either the mean hourly or the cumulative value across each period
(see legend of Table 1 for precise definition of each Outcome). Sleep bouts are analyzed
on the log scale to stabilize the variation, all other variates are analyzed on the linear
scale. Each outcome in each period is analyzed by analysis of covariance using treatment
group and treatment date as factors and the corresponding pre-treatment interval, 24 hrs
earlier, as the covariate. Adjusted means and the change from vehicle means and their
corresponding standard errors are summarized for each treatment group. Outcomes
analyzed on the log scale are back-transformed to report geometric means and mean ratioto-
vehicle results.
The compound of Examples 3 is tested essentially as described. The compound of
Example 3 is found to significantly increase cumulative NREM sleep time and
cumulative total sleep time without significant rebound insomnia, REM sleep inhibition
or inhibition of locomotor intensity (LMI) at 3 mg/kg. (See sleep profile and locomotor
activity intensity in Table 2.)
C p mi- E *
Efficacv variables Undesired effect variables
Cumulative NREM sleep Rebound Insomnia
Dose (mg/kg PO) N Adj. Mean SE N Adj. Mean LCL
10 9 29.1 6.4 9 -3.1 -11.0
3 10 31.7 6.1 10 0.3 -7.3
1 4 34.0 9.0 4 1.6 -9.8
0.5 4 32.9 9.0 4 -5.0 -16.3
0.25 12 37.9 6.0 12 -2.2 -9.8
0.1 8 26.7 7.4 8 -3.7 -13.0
0.05 14 23.4 5.7 14 0.2 -7.0
0.035 7 19.2 7.1 7 1.3 -7.7
0.025 10 6.5 6.1 10 1.4 -6.3
Cumulative Total sleep REM inhibition
Dose (mg/kg PO) N Adj. Mean SE N Adj. Mean LCL
10 9 26.5 7.1 9 -9.4 -3.8
3 10 29.7 6.8 10 -4.6 0.7
1 4 33.6 10.1 4 -0.1 7.8
0.5 4 30.9 10.1 4 0.4 8.2
0.25 12 38.9 6.7 12 2.0 7.2
0.1 8 28.7 8.3 8 4.9 11.4
0.05 14 25.3 6.5 14 4.2 9.2
0.035 7 18.9 8.0 7 -1.8 4.4
0.025 10 7.7 6.9 10 0.5 5.8
Average Sleep Bout Locomotor Activity Intensity
Dose (mg/kg PO) N Adj. Mean SE N Adj. Mean LCL
10 9 2.0 0.2 9 -1.9 -0.1
3 10 2.0 0.2 10 -3.1 -1.3
1 4 1.7 0.3 4 -1.6 0.9
0.5 4 1.8 0.3 4 0.6 3.1
0.25 12 2.1 0.2 10 -0.7 1.1
0.1 8 1.7 0.2 8 0.1 2.3
0.05 14 1.6 0.2 10 -0.3 1.6
0.035 7 1.3 0.2 6 0.2 2.4
0.025 10 1.2 0.1 9 0.2 2.1
Longest Sleep Bout
Table 2. Outcome statistics: Abbreviations: N = sample size; Adj.Mean = adjusted
group mean value relative to vehicle controls; SE = standard error of
the mean; LCL = lower 95% confidence limit, NREM = non-REM, i.e.,
all sleep other than REM sleep. The parallel reference vehicle group
sample size was N=21.
Definitions and units — means are adjusted differences from vehicle controls:
• Cumulative sleep: across the first 6 hr. post-treatment, in minutes ('Total sleep'
denotes NREM sleep + REM sleep).
• Average sleep bout: average of hourly-averaged sleep bouts, across the first 6 hr.
post-treatment, expressed as w-fold increase over vehicle controls.
• Longest sleep bout: the longest sleep bout in the first 6 hr. post-treatment,
expressed as w-fold increase over vehicle controls.
• Rebound insomnia: cumulative minutes of NREM+REM sleep during the first 3
hr. of the lights on period, i.e., 7th, 8th and 9th hours post-treatment.
• REM inhibition: cumulative minutes of REM sleep during the first 12 hr. posttreatment.
• Locomotor Activity (LMA) Intensity: expressed as LMA counts per minute of
EEG-defined wakefulness, averaged across the first 6 hr. post-treatment.
Determining efficacy. The threshold efficacy for each of the four efficacy variables is
calculated by plotting the increase in each variable relative to vehicle controls during the
6 hr. period after treatment against log(dose). The threshold efficacy for each variable is
that dose, estimated by 4 paramater logistic nonlinear regression, which gives the defined
efficacy threshold value; +30 min. of additional accumulated non-REM sleep, +25 min.
of additional accumulated total sleep, 1.75x increase in average sleep bout duration, and
1.5x increase in longest sleep bout duration. The compound of example 3 is found to
have threshold efficacious doses as shown in Table 3.
Estimated efficacy 95% confidence
Table 3 dose (mg/kg) interval (mg/kg)
NREM accumulation = 30 min. 0.09 0.04-n.e.*
total sleep accumulation = 25 min. 0.05 0.03-0.09
longest sleep bout (1.75 -fold increase) 0.12 0.07-0.20
average sleep bout (1.5-fold increase) 0.04 0.03-0.06
*n.e. = not estimable, statistically.
. 1
Dose {mg / kg }
Rebound
In s omnia
(minutes)
0.01 0 .1 10
Dose (mg/kg)
Dos f g kg
Determining undesired effects. Each 'undesired effect' outcome variable (see Table 2
legend for definitions), is plotted against log(dose). The threshhold value for REM
inhibition is defined as a cumulative reduction of REM sleep of -10 min. The threshold
value for rebound insomnia is defined as -20 min. The threshold value for reduced LMI
is defined as -5 locomotor activity counts per minute of EEG-defined wakefulness. A
significant undesired effect is defined to occur when the lower confidence limit goes
below the threshold value at any dose at or below 10 times the average efficacious dose,
and a dose response trend is evident for doses above the threshhold efficacy dose. For
the compound of Example 3, no undesired occurences of REM inhibition, rebound
insomnia, or reduction in LMI are observed at doses up to at least 1.2 mg/Kg (1.2 mg/Kg
is 10 times the most conservative efficacy dose of 0.12 mg/kg [Table 3]). Negative
values indicate REM inhibition, rebound insomnia and reduced LMI, respectively.
In further studies, the compounds of the present invention may be coadministered
with selective serotonin reuptake inhibitors to demonstrate a potentiation of their effect on
non-rapid eye movement sleep (NREM sleep) and sleep maintenance. The compound of
Example 3 is coadministered with citalopram in rat sleep studies essentially as described
above for the compound alone, and is found to significantly increase NREM sleep at
significantly lower doses.
Plasma Clearance: It is important in a compound useful for treating sleep disorders such
as insomnia that it be adequately cleared from the body with a favorable rate of clearance
to avoid unwanted effects such as prolonged somnolence beyond the desired sleep period,
daytime sleepiness, impaired cognition after waking, etc. The present invention provides
compounds with improved rates of clearance. Rate of clearance can be assayed
essentially as described below.
Male Sprague Dawley rats (body weight range 250-320 g) with indwelling
femoral arterial cannulae are obtained from Charles River, Wilmington, MA 01887, USA.
Test compound is administered intravenously in solution ( 1 mL/kg) in 20% Captisol® in
22.5 mM phosphate buffer, pH 2, at a final drug concentration of 1.0 mg/mL (free base
equivalents). Blood samples are obtained using the indwelling cannula over 24 hr.
Samples of plasma are obtained by centrifugation and stored frozen (-20°C), or on dry ice,
prior to analysis.
Male Beagle dogs (body weight range 10-12 kg) are obtained from Marshall
Bioresources, USA. Test compound is administered intravenously in solution ( 1 mL/kg)
in 20% Captisol ® in 22.5 mM phosphate buffer, pH 2, at a final drug concentration of 1.0
mg/mL (free base equivalents). Blood samples are obtained from the jugular vein over
24 hr. Samples of plasma are obtained by centrifugation and stored frozen (-20°C) prior
to analysis.
Frozen plasma samples are thawed to room temperature for bioanaly
concentrations of test compound. A related internal standard compound in acetonitrile/
methanol (1:1, v/v) is added to all samples of plasma (1:1, v/v). The samples are
centrifuged to remove precipitated protein prior to analysis. The supernatants are
analysed by injection and rapid gradient elution on a Javelin Betasil CI 8 column (20 x 2.1
mm cartridge, Mobile phase A: Water/ 1M NH4HCO3 , 2000: 10 v/v, Mobile Phase B:
MeOH/ 1M NH4HCO3 , 2000: 10 v/v). The eluted analytes are detected by LC-MS-MS
analysis using a Sciex API 4000 triple quadrupole mass spectrometer. Concentrations of
compound are determined from standards prepared and analysed under identical
conditions. Clearance is calculated using non-compartmental analysis in Watson 7.4,
Thermo Fisher Scientific, Inc.
Clearance = Dose
Area under curve for plasma concentration / time
The compounds of Examples 1 and 3 are run essentially as described and are
found to have favorable clearance profiles:
Example Clearance (mL/min./Kg)
Rat Dog
1 14.1 (+/- 6.9, n=3)
3 14.5 (+/- 1.4, n=3) 3.2 (+/- 0.7, n=3)
While it is possible to administer the compounds as employed in the methods of
this invention directly without any formulation, the compounds are usually administered
in the form of pharmaceutical compositions comprising the compound, or a
pharmaceutically acceptable salt thereof, as an active ingredient and at least one
pharmaceutically acceptable carrier, diluent and/or excipient. These compositions can be
administered by a variety of routes including oral, sublingual, nasal, subcutaneous,
intravenous, and intramuscular. Such pharmaceutical compositions and processes for
preparing them are well known in the art. See, e.g., Remington: The Science and Practice
of Pharmacy (University of the Sciences in Philadelphia, ed., 2 1st ed., Lippincott
Williams & Wilkins Co., 2005).
The compositions are preferably formulated in a unit dosage form, each dosage
containing from about 0.1 to about 60 mg, more usually about 0.5 to about 30 mg, as for
example between about 1 and about 10 mg of the active ingredient. The term "unit
dosage form" refers to physically discrete units suitable as unitary dosages for human
subjects and other mammals, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect, in association with at least
one suitable pharmaceutically acceptable carrier, diluent and/or excipient.
The compounds of Formula I are generally effective over a wide dosage range.
For example, dosages per day normally fall within the range of about 0.002 to about 1.0
mg/kg, more usually from about 0.008 to 0.5 mg/kg, and as for example between 0.015
and 0.15 mg/kg of body weight. In some instances dosage levels below the lower limit of
the aforesaid range may be more than adequate, while in other cases still larger doses may
be employed without causing any harmful side effect, and therefore the above dosage
ranges are not intended to limit the scope of the invention in any way. It will be
understood that the amount of the compound actually administered will be determined by
a physician, in the light of the relevant circumstances, including the condition to be
treated, the chosen route of administration, the actual compound or compounds
administered, the age, weight, and response of the individual patient, and the severity of
the patient's symptoms.
We Claim:
1. A compound of the formula
or a pharmaceutically acceptable salt thereof.
2. A compound according to Claim 1 which is an HC1 salt.
3. A pharmaceutical composition comprising a compound according to Claim 1, or a
pharmaceutically acceptable salt thereof, in combination with at least one
pharmaceutically acceptable carrier, diluent, or excipient.
4. A method of treating insomnia in a mammal comprising administering to a mammal in
need of such treatment an effective amount of a compound according to Claim 1, or a
pharmaceutically acceptable salt thereof.
5. The method of Claim 4 where the mammal is a human.
6. The method of Claim 4 where the insomnia is characterized by difficulties in sleep
onset or sleep maintenance or both.
7. The method of Claim 6 where the mammal is a human.
8. A compound according to Claim 1, or a pharmaceutically acceptable salt thereof, for
use in therapy.
9. A compound according to Claim 1, or a pharmaceutically acceptable salt thereof, for
use in the treatment of insomnia.
10. The compound for use according to Claim 9, or a pharmaceutically acceptable salt
thereof, where the insomnia is characterized by difficulties in sleep onset or sleep
maintenance or both.
11. The compound for use according to either Claim 9 or 10 in a human.
12. The use of a compound according to Claim 1, or a pharmaceutically acceptable salt
thereof, in the manufacture of a medicament for the treatment of insomnia.
13. The use according to Claim 12, where the insomnia is characterized by difficulties in
sleep onset or sleep maintenance or both.
14. A pharmaceutical composition comprising a compound according to claim 1, or a
pharmaceutically acceptable salt thereof, in combination with at least one
pharmaceutically acceptable carrier, excipient or diluent, and optionally other therapeutic
ingredients.
15. The pharmaceutical composition of Claim 14 where the other therapeutic ingredient
comprises a selective serotonin reuptake inhibitor.