(THIENO[2,3-b] [1,5]BENZOXAZEPIN-4-YL)PIPERAZIN-1-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 H1/5-HT2Ainverse agonists/antagonists are currently commercially
available.
WO 2007/022068 describes certain substituted (thieno[2,3-b][1,5]benzodiazepine-
4-yl)piperazin-1-yl and (thieno[2,3-b][1,5]benzoxazepine-4-yl)piperazin-1-yl compounds
for treating sleep disorders.
The present invention provides 3-[4-(2,8-dimethyl-thieno[2,3-
b][1,5]benzoxazepin-4-yl)piperazin-1-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
or a pharmaceutically acceptable salt thereof. That is to say 3-[4-(2,8-dimethylthieno[2,3-
b][1,5]benzoxazepin-4-yl)piperazin-1-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:
"Brine" means saturated aqueous NaCl solution.
"DMEM" means Dulbecco's Minimum Eagle's Medium.
"DMSO" means dimethyl sulfoxide.
"EDTA" means ethylenediaminetetraacetic acid.
"Equiv" means equivalent(s).
"FBS" means fetal bovine serum.
"HEPES" means 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid.
"HPLC" means high pressure liquid chromatography
"hr." means hour or 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 minute or minutes.
"MS" means mass spectroscopy.
"MS (ES+)" means mass spectroscopy using electrospray ionization.
"NMR" means nuclear magnetic resonance.
"RO" means receptor occupancy.
"THF" means tetrahydrofuran.
General Chemistry
The compound of the present invention can be prepared according to the
following synthetic examples.
Preparation 1: Methyl 5-methylthiophene-3-carboxylate
Combine 4-bromo-2-methyl-thiophene (24.5 g, 138.37 mmol), bis-(1,3-
diphenylphosphino)propane (2.35 g, 5.53 mmol), palladium acetate (1.24 g, 5.53 mmol),
triethylamine (38.57 mL, 276.73 mmol), methanol (40 mL) and dimethyl sulphoxide (80
mL) and purge the reaction vessel with carbon monoxide. Pressurize to 30 PSI with
carbon monoxide and heat to 70°C, with stirring overnight under carbon monoxide.
Evaporate the volatiles under reduced pressure to give a dark orange solution. Add water
(300 mL) and extract with ethyl acetate (3 x 150 mL) then wash the combined organic
extracts with water (2 x 100 mL). Dry over sodium sulphate, filter and evaporate under
reduced pressure. Add 10% diethyl ether in iso-hexane (500 mL) and remove the
resulting dark yellow precipitate by filtration. Evaporate the filtrate under reduced
pressure to give methyl 5-methylthiophene-3-carboxylate (18.894 g, 87.42% yield) as a
liquid. 1H MR (400.13 MHz, CDC13) : 7.84 (d, J = 1.5, 1H), 7.16 (t, J = 1.5, 1H), 3.83
(s, 3H) and 2.46 (d, J =1.5, 3H).
Preparation 2. 5-Methylthiophene-3-carboxylic acid
To a 250 mL flask add methyl 5-methylthiophene-3-carboxylate (16.4 g,
104.99 mmoles, 1.00 equiv) and dissolve in methanol (57.40 mL). Add NaOH (12.60 g,
314.97 mmoles, 3 equiv) and water (19.68 mL). Heat the suspension at reflux (65°C) for
45 min., until the reaction is complete by LC-MS. Cool the reaction mixture to room
temperature, then add 5M aqueous HCl (85 mL, 5vol), followed by ethyl acetate
(130 mL, 7.5vol). Stir for 15 min., until all solids are dissolved. Separate the layers and
extract the aqueous layer with ethyl acetate (2 x 130 mL). Wash the combined organics
with water (3 x 130 mL), then brine (85 mL). Dry over sodium sulfate, filter and
concentrate to give a solid of 5-methylthiophene-3-carboxylic acid (13.93 g, 93.32%
yield). MS (m/z): 141.0 (M-H). 1H NMR (400.13 MHz, CDC13) : 7.99 (d, J= 1.5 Hz,
1H), 7.20 (d, t= 1.5 Hz, 1H), 2.50 (d, J = 1.5, 3H).
Preparation 3. 2-Bromo-5-methyl-thiophene-3-carboxylic acid
To a solution of 5-methyl-thiophene-3-carboxylic acid (13.93 g, 97.98 mmoles,
1.00 equiv) in acetic acid ( 119.80 mL), add bromine (5.03 mL, 97.98 mmoles, 1.00 equiv)
dropwise as a solution in acetic acid (59.90 mL) over 20 min. Stir the reaction mixture at
room temperature for 1 hr., until the reaction is complete by LC-MS. Pour the reaction
mixture slowly into water (696.50 mL) at room temperature, and collect the resulting
precipitate by filtration. Wash with water (100 mL) and dry under vacuum and nitrogen
on a pad for 16 hr. to give a white solid of 2-bromo-5-methyl-thiophene-3-carboxylic acid
(20.55 g, 94.88% yield). MS (m/z): 218.89, 220.79 (M-H). 1H NMR (400.13 MHz,
CDC13) : 7.08 (d, J = 1.5 Hz, 1H), 2.42 (s, 3H).
Preparation 4. 2-Bromo-N-(2 -hydroxy -4-methyl-phenyl)-5-methyl-thiophene-3-
carboxamide
To a suspension of 2-bromo-5-methyl-thiophene-3-carboxylic acid (45.2 g, 204.46
mmoles, 1.00 equiv) in dichloromethane (3 16.40 mL) in a 1 L flask, add
dimethylformamide (500 ), followed by 2M oxalyl chloride in dichloromethane
( 112.45 mL, 224.90 mmoles, 1.1 equiv) dropwise (Caution gas evolution). Stir the
reaction mixture at room temperature for 2 hr., until the reaction mixture becomes a clear,
dark solution and the reaction is complete by TLC (eluent EtOAc 30% in iso-hexane).
Evaporate the solution to dryness and then redissolve the resulting solid in anhydrous
tetrahydrofuran (678 mL). Add this solution to a flask containing 6-amino-m-cresol
(33.40 g, 265.79 mmoles 1.3 equiv), pyridine (82.67 mL, 1.02 moles, 5 equiv) and
anhydrous tetrahydrofuran (678 mL). Stir at room temperature for 2 hr. until the reaction
is complete by LC-MS. Pour the reaction mixture into 1M aqueous HCl (2.71 L, 60 vol)
with stirring and collect the resulting precipitate by filtration. Wash with 1M aqueous
HCl (250 mL), then water (250 mL), and dry under vacuum on a filter pad overnight to
give a red solid 2-bromo-N-(2-hydroxy-4-methyl-phenyl)-5-methyl-thiophene-3-
carboxamide (62.34 g; 93.47% yield). If necessary, the solid can be purified further by
washing with methanol. MS (m/z): 325.98, 327.93 (M+H).
- -
ln a 500 mL flask dissolve 2-bromo-N-(2-hydroxy-4-methyl-phenyl)-5-methylthiophene-
3-carboxamide (62.34 g, 191.10 mmoles, 1.00 equiv) in dimethyl sulfoxide
(1.56 L) and add potassium carbonate (79.23 g, 573.30 mmoles, 3 equiv). Heat the
reaction mixture to 140°C with stirring for 4 hr., until the reaction is complete by LC-MS.
Cool the reaction mixture to room temperature and then pour into water (2.5 L)
controlling the exotherm with an ice bath. Acidify with 2M aqueous HCl (450 mL),
extract with dichloromethane (4 x 800 mL) and wash the organic phase with saturated
aqueous sodium bicarbonate (1.2 L), then water (2 x 2 L). Dry, filter and concentrate.
Purify by flash chromatography on silica gel (750 g) eluting with 0% to 20% ethyl acetate
in dichlromethane to give a solid of 2,8-dimethyl-5H-thieno[2,3-b][1,5]benzoxazepin-4-
one (28.1 g; 59.94%). MS (m/z): 246.09 (M+H).
Preparation 6. 4-Chloro-2,8-dimethyl-thieno[2,3-b][1,5]benzoxazepine
Heat a suspension of 2,8-dimethyl-5H-thieno[2,3-b][1,5]benzoxazepin-4-one
(26.84 g, 109.42 mmoles, 1.00 equiv) in methoxybenzene (107.36 mL) in a 500 mL flask
to about 60°C. Add N,N-dimethylaniline (38.89 mL, 306.37 mmoles, 2.8 equiv) in one
portion, followed by phosphoryl chloride (23.39 mL, 251.66 mmoles, 2.3 equiv) over 15
min. Heat the resulting solution at 100°C for 2 hr., until the reaction is complete by LCMS.
Cool the reaction mixture slightly and evaporate to give a dark oil of 4-chloro-2,8-
dimethyl-thieno[2,3-b][1,5]benzoxazepine (28.85g, assumed quantitative). MS (m/z):
264.0 (M+H).
Preparationo 7. Methyl 3-[4-(2,8-dimethylthieno[2,3-b][1,5]benzoxazepin-4-
yl)piperazin-1-yl]-2,2-dimethyl-propanoate
To a solution of 4-chloro-2,8-dimethyl-thieno[2,3-b][1,5]benzoxazepine (28.85 g,
109.39 mmoles, 1.00 equiv) in acetonitrile (288.50 mL), add methyl 2,2-dimethyl-3-
piperazin-1-yl-propanoate (59.77 g, 218.77 mmoles , 2 equiv) and potassium carbonate
(136.06 g, 984.47 mmoles, 9 equiv). Heat the mixture at a gentle reflux (82°C) for 3 hr.,
until the reaction is complete by LC-MS. Cool the reaction and evaporate. Then partition
between water (300 mL) and ethyl acetate (3 x 300 mL). Wash the combined organic
extracts with water (2 x 300 mL), then brine. Dry over sodium sulfate, filter and
evaporate. Purify by flash chromatography on silica gel (800 g) eluting with 0% to 30%
ethyl acetate in iso-hexane to give a thick oil of methyl 3-[4-(2,8-dimethylthieno[2,3-
b][1,5]benzoxazepin-4-yl)piperazin-1-yl]-2,2-dimethyl-propanoate (37.95 g; 81.14%
yield). MS (m/z): 428.1 (M+H).
Example 1. 3-[4-(2,8-Dimethylthieno[2,3-b][1,5]benzoxazepin-4-yl)piperazin-1-yl]-2,2-
dimethyl-propanoic acid
To a slurry of methyl 3-[4-(2,8-dimethylthieno[2,3-b][1,5]benzoxazepin-4-
yl)piperazin-1-yl]-2,2-dimethyl-propanoate (37.95 g, 88.76 mmoles, 1.00 equiv) in
isopropyl alcohol (227.70 niL) and water (227.70 mL), add sodium hydroxide (10.65 g,
266.27 mmoles, 3 equiv) and heat the mixture to gentle reflux (85°C) for 1.5 hr., until
complete dissolution occurs and the reaction is complete by LC-MS. Cool to room
temperature and neutralize to around pH 6.0 - 6.5 using 2M aqueous HCl (approx. 120
mL), to give a precipitated solid. Remove the organic solvent in vacuo to give a
suspension. Check the pH of the suspension and if necessary further adjust to pH 6.0-6.5
with 2M aqueous HCl (approx. 15 mL). Filter the suspension and wash with water, then
dry overnight under vacuum. Slurry the crude solid material in acetonitrile (100 mL,
2.5vol), sonicate, and then gently heat (40°C) with stirring for 15 min. Filter, wash with
acetonitrile (3 x 20 mL), then dry the product under vacuum to give a solid of 3-[4-(2,8-
dimethylthieno[2,3-b][1,5]benzoxazepin-4-yl)piperazin-1-yl]-2,2-dimethyl-propanoic
acid (29.5 g, 80.37% yield). MS (m/z): 414.1 (M+H). 1H NMR (400.13 MHz, CDC13) :
6.99 (d, J = 7.8 Hz, 1H), 6.90 (dd, J = 7.8 and 1.5 Hz, 1H), 6.83 (d, J = 1.5 Hz, 1H), 6.28
(s, 1H), 3.67 (br s, 4H), 2.86 (br s, 4H), 2.59 (s, 2H), 2.34 (s, 3H), 2.28 (s, 3H) 1.26 (s,
6H)
Example 2. 3-[4-(2,8-Dimethylthieno[2,3-b][1,5]benzoxazepin-4-yl)piperazin-1-yl]-2,2-
dimethyl-propanoic acid dihydrochloride
To a suspension of 3-[4-(2,8-dimethylthieno[2,3-b][1,5]benzoxazepin-4-
yl)piperazin-1-yl]-2,2-dimethyl-propanoic acid (300 mg, 725.44 , 1.00 equiv) in
isopropyl alcohol (4.50 mL) at 50°C, add 4M HCl in 1,4-dioxane (453.40 , 1.81
mmoles, 2.5 equiv). Heat the resulting solution to 60°C for 30 min., then cool to room
temperature. Evaporate under vacuum, slurry the resulting solid in diethyl ether (4.50
mL) for 15 min., then collect by filtration, and dry on the pad. Further dry in vacuum
oven for 1 hr. to give a crystalline solid of 3-[4-(2,8-dimethylthieno[2,3-
b][1,5]benzoxazepin-4-yl)piperazin-1-yl]-2,2-dimethyl-propanoic acid dihydrochloride
(346 mg, 98.0%). MS (m/z): 414.0 (M+H).
Example 3. 3-[4-(2,8-Dimethylthieno[2,3-b][1,5]benzoxazepin-4-yl)piperazin-1-yl]-2,2-
dimethyl-propanoic acid; 4-methylbenzenesulfonic acid
To a suspension of 3-[4-(2,8-dimethylthieno[2,3-b][1,5]benzoxazepin-4-
yl)piperazin-1-yl]-2,2-dimethyl-propanoic acid (300 mg, 725.44 , 1.00 equiv) in
ethanol (6 mL) at 50°C, add p-toluenesulfonic acid (124.92 mg, 725.44 , 1 equiv)
and stir the resulting solution at 50°C for 30 min. Cool to room temperature and
evaporate under vacuum to give a solid. Slurry the solid in diethyl ether (4.50 mL) for 15
min., then collect by filtration, and dry on the pad. Further dry in vacuum oven for 1 hr.
to give a crystalline solid of 3-[4-(2,8-dimethylthieno[2,3-b][1,5]benzoxazepin-4-
yl)piperazin-1-yl]-2,2-dimethyl-propanoic acid; 4-methylbenzenesulfonic acid (413 mg,
97%). MS (m/z): 414.0 (M+H).
Literature data (Morairty SR, Hedley L, Flores J, Martin R, Kilduff TS. (2008)
Selective 5-HT2Aand 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 WakeDisorders, CNS & Neurological
Disorders - Drug Targets, vol 6, pg. 31-43 (2007)) and data generated in non-clinical
animal studies support a role for dual activity HI inverse agonists / 5-H T 2A 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 g 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
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 g 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 1-(1-
Naphthyl) piperazine. Samples are incubated for 4 hr. at room temperature (22° C) and
then read in a Microbeta Trilux.
5-HT2C 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 g membranes to assay buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM EDTA,
10 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 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 (Ki ) are calculated according to the equation Ki =
IC50/(l+L /Kd), where L equals the concentration of radioligand used in the experiment
and Kd equals the equilibrium dissociation constant of the radioligand for the receptor,
determined from standard saturation analysis or homologous competition experiments.
Reported values for Ki , 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 Ki 1
+ log Ki 2 +. . .log Ki 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 E l 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
(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 ) . 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-HT 1B, 5-HT 1D, receptors, lack of agonist activity at 5-HT2Breceptors, 5-
HT2c, 5-HT , 5-HT , and 5-HT 7 receptors), dopaminergic receptors (specifically Dl, D2,
and D3), GABAA receptors, adrenergic receptors and monoamine transporters.
The compound of example 2 is tested essentially as described above and is found
to have activity profiles as shown in Table 1.
Table 1. Selectivity data
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-HT2C 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-HT2A Receptor Occupancy: Receptor occupancy (RO) is assayed to demonstrate
antagonist/inverse agonist activity at the 5-HT2A Receptor in vivo. Briefly, male Sprague-
Dawley rats (Harlan Sprague-Dawley, Indianapolis, ) 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)-1-[2-(4-fluorophenyl)ethyl]-4-
piperidinemethanol), a selective 5-HT2Aantagonist, is used as a tracer. MDL 100907 is
suspended in water with 5 ΐ /F2 10.87 dilute lactic acid ( 1 mg/ml), diluted to 6 g/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 g kg. Rats are administered test compound, ketanserin, or vehicle (N =
4), followed 1 hr. later with an intravenous, 3 g/kg tracer dose of MDL 100907. It is at
the time of tracer administration that 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 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 2 is tested essentially as
described and is found to achieve high 5-H T2A receptor occupancy with an ED 0 of 0.09
mg/kg.
Inhibition of DPI 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-H T 2A 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 subcutaneously,
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 2 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 RN, 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 ( I0,000, 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 2 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 Example 2 is tested essentially as described. The compound of
Example 2 is found to significantly increase cumulative NREM sleep time and
cumulative total sleep time without significant rebound insomnia, or inhibition of
locomotor intensity (LMI) at 3 mg/kg. REM sleep inhibition, however, cannot be ruled
out by these data. (See sleep profile and locomotor activity intensity in Table 2.)

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 2 is found to
have threshold efficacious doses as shown in Table 3.

legend for definitions), is plotted against log(dose). The threshhold value for REM sleep
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 2, REM sleep inhibition exceeded the threshhold value at 3
mg/kg, but this dose is more than 10 times the most conservative efficacy dose of 0.26
mg/kg (Table 3); apparent REM sleep inhibition at 0.25 mg/kg and 0.025 mg/kg are not
part of a dose related trend and are not statistically significant at those doses, and may
therefore be considered an artifact of the small sample size at those doses; nevertheless,
REM sleep inhibition cannot be ruled out by these data. It is concluded that no undesired
occurences of REM rebound insomnia, or reduction in LMI are observed within 10 times
the most conservative efficacy dose, but REM sleep inhibition cannot be ruled out.
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 2 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 bioanalysis of
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.
The compound of Example 2 is run essentially as described and is found to have a
favorable clearance profile:
Example Clearance (mL/min./Kg)
Rat Dog
2 27 (+/- 7.3, n=3) 2.8 (+/- 1.2, 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., 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 HCl salt.
3. A compound according to Claim 1 which is a tosylate salt.
4. 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.
5. A method of treating insomnia in a mammal comprising administering to a mammal
need of such treatment an effective amount of a compound according to Claim 1, or a
pharmaceutically acceptable salt thereof.
6. The method of Claim 5 where the mammal is a human.
7. The method of Claim 5 where the insomnia is characterized by difficulties in sleep
onset or sleep maintenance or both.
8. The method of Claim 7 where the mammal is a human.
9. A compound according to Claim 1, or a pharmaceutically acceptable salt thereof, for
use in therapy.
10. A compound according to Claim 1, or a pharmaceutically acceptable salt thereof, for
use in the treatment of insomnia.
11. The compound for use according to Claim 10, or a pharmaceutically acceptable salt
thereof, where the insomnia is characterized by difficulties in sleep onset or sleep
maintenance or both.
12. The compound for use according to either Claim 10 or 11 in a human.
13. 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.
14. The use according to Claim 13, where the insomnia is characterized by difficulties in
sleep onset or sleep maintenance or both in a mammal.
15. 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.
16. The pharmaceutical composition of Claim 15 where the other therapeutic ingredient
comprises a selective serotonin reuptake inhibitor.