Specification
FIELD OF THE INVENTION
The present invention provides compounds of Formula (I), and a composition thereof, used as
allosteric potentiation of the M4 subtype of muscarinic receptor.
BACKGROUND OF THE INVENTION
The present invention provides compounds that are selective allosteric modulators ot the M
subtype of muscarinic receptor. The M4 muscarinic receptor is believed to play a role in modulating
synaptic function in key areas of the brain involved in regulating mood, cravings, attention and
cognition. As a result, it provides a novel therapculic target tor the treatment oi' psychosis; attention
disorders, such as attention deficit hyperactivity disorder (ADHD): cognitive disorders, including
memory loss; and drug addiction. The M2 and M4 subtypes of muscarinic receptor are also involved in
muscarinic agonist- induced analgesic effects, but it is believed that side effects of such treatment are
associated primarily with M2 receptor activation. Thus, compounds that selectively modulate M4
receptors would provide a novel treatment strategy for neuropathic pain, without unwanted side
e ffeet s.
Unlike compounds that act at the neurotransmitter binding site (orthosteric site), allosterie
modulators act at a distinct site on the receptor. The use of allosteric modulators provides several
advantages in the treatment of disease. Christoponlos Nature Reviews (2002) 1:19X-210. For instance,
under saturating conditions (high concentrations of allosteric potentiator) one would not expect
excessive stimulation of the M4 muscarinic receptor, since it is dependent on the endogenous
neurotransmitter for activation. Second, allosteric agonists exert their physiological effects only in the
presence of endogenous agonist. As a result, allosteric potentiators are less likely to produce the
condition of receptor desensitzation or down-regulation that are associated with excessive cholinergic
stimulation, finally, allosteric modulators are likely to show greater receptor selectivity, especially as
the orthosteric binding site is well conserved between muscarinic receptor subtypes.
At present, no selective allosteric modulators of the M4 subtype of muscarinic receptor have
been reported. The development of selective M4 allosteric potentiators will therefore greatly enhance
the ability to treat disorders such as psychosis and pain, without unwanted side effects. Thas, the
present invention provides a class of allosteric. modulators of M4 muscarinic receptors, compositions
comprising these compounds, and methods of using the compounds.
BRIEF SUMMARY OF THE INVENTION
The invention provides compounds of formula (I):
n is 0. I, or 2:
X is a bond. -O-. -SCp-C(O)-, -NR'X -C(0)-KR2-. or -NR2-C(0>:
p is 0. 1 , or 2:
R is hydrogen, hydroxyl. CVO alkyl. phenyl, pyridyl, pyrrolidinyi. pipcra/dnyl. morphol no.
thiazolyF imida/'oivl, or I ..i-dinxalaiiyl: ^vhieh phenyl, pipera/.inyl, or tnla/ulyl group may be
optionally substituted with one substiluenl selected from the group consisting of halo or d-C' alkyl;
wherein n cannot be 0 when p is 0. or when X is -<)-, -KR\ or -r\R2-ClO)-:
R* is hydrogen or C|-C> alkyl:
which C'i-C: alkyl may be optionally substituted with one hydroxyl; or a pharmaceutically acceptable
salt thereof.
The compounds of Formula I are muscarinic receptor potentiators. Specifically, the
compounds of formula 1 are allosleric potentiators of the M4 subtype of muscarinic receptor. Because
these compounds potentiate the physiological effects associated with M.( receptor activation, the
compounds arc useful in the treatment of disorders related 10 inadequate Mj receptor activation, fhese
disorders include: pyschosis (particularly, schizophrenia): cognitive disorders (for example, memory
loss); attention disorders (such as attention deficit hyperactivity disorder); and pain (in particular,
neuropathic pain).
In one embodiment, this invention provides a pharmaceutical composition comprising, as an
active ingrethent, a compound of Formula 1. or a pharmaceutically acceptable salt thereof, in
combination with one or more pharmaceutically acceptable carriers, diluents, or excipients. hi 60g, 7.0mmol) in THF (3ml) at room
temperature is added dropwiss a solution of lithium bis(trimethylsilyl)amide (1.0M in
hexanes)(3.50ml, 3.50mmol). The reaction mixture is stirred for 15 minutes and then is
treated with 3-amino-5-chloro-6-rnethanesulfmyl-4-methyI-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide (0.345g, l.OOmmol). The reaction mixture is heated at
75°C in a sealed tube for 1.5 hours, cooled to room temperature, and then quenched by
the addition of water (10ml). The reaction mixture is transferred to an erlenmeyer flask
and diluted to 75ml with water. The mixture is cooled to 0°C and stirred. During this
time, a yellow precipitate is formed. This solid is collected by filtration and slurried in a
2:1 EtOAc:hexane mixture. This slurry is stirred for 10 minutes, filtered, and the
collected solid is placed in the vacuum oven to dry (60°C for 16 hours). This affords the
title compound as a yellow solid (0.242g, 58%). Mass (m/z): 417.2 (M++l), 415.2 (IvT-1).
Example 20
3-Amino-5-chloro-4-methyl-6-(2-pyridin-4-yl-ethoxy)-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide
To a solution of 4-pyridineethanol (0.862g, 7.0mmol) in THF (3ml) at room
temperature is added dropwise a solution of lithium bis(trimethylsilyl)amide (1.0M in
hexanes)(3.50ml, 3.50mmol). The reaction mixture is stirred for 15 minutes and then is
treated with 3-amino-5-chloro-6-methanesulfinyl-4-methyl-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide (0.345g, l.OOmmol). The reaction mixture is heated at
75°C in a sealed tube for 1,5 hours, cooled to room temperature, and then quenched by
the addition of water (10ml). The reaction mixture is transferred to an erlenmeyer flask
and diluted to 75ml with water. The mixture is cooled to 0°C and stirred. During this
time, a yellow precipitate is formed. This solid is collected by filtration and slurried in a
2:1 EtOAc:hexane mixture. This slurry is stirred for 10 minutes, filtered, and the
collected solid is placed in the vacuum oven to dry (60°C for 16 hours). This affords the
title compound as a yellow solid (0.23 lg, 57%). Mass (m/'z): 403.1 (M++l), 401.1 (M+-1).
Example 21
3-Amino-6-{2-[bis-(2-hydroxyethyl)-amino]-ethoxy}-5-chloro-4-methyl-thieno[2,3-
b]pyridine-2-carboxylic acid cyclopropylamide
To a solution of triethanolamine (1.044g, 7.0mmol) in THF (3ml) at room
temperature is added dropwisea solution of lithium bis(trimethylsilyl)amide (1.0M in
hexanes)(3.50ml, 3.50mmol). The reaction mixture is stirred for 15 minutes and then is
treated with 3-amino-5-chloro-6-methanesulfinyl-4-mcthyl-thieno[2,3-b]pyridinc-2-
carboxylic acid cyclopropylamide (0.345g, l.OOmmol). The reaction mixture is heated at
75CC in a sealed tube for 1.5 hours, cooled to room temperature, and then quenched by
the addition of water (10ml). The reaction mixture is transferred to an erlenmeyer flask
and diluted to 75ml with water. The mixture is cooled to 0°C and stirred. During this
time, a yellow precipitate is formed. This solid is collected by filtration and slurried in a
2:1 EtOAc: hexane mixture. This slurry is stirred for 10 minutes, filtered, and the
collected solid is placed in the vacuum oven to dry (60°C for 16 hours). This affords the
title compound as a yellow solid (0.179g, 41%). Mass (rn/z): 429.2 (M++l), 427.2 (M+-l).
Example 22
3-Amino-5-chloro-6-([l,3]clioxolari-4-ylmethoxy)-4rmethyl-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide
To a solution of glycerol formal (0.729g, 7.0mmol) in THF (3ml) at room
temperature is added dropwise a solution of lithium bis(trimethylsilyl)amide (1.0M in
hexanes)(3.50ml, 3.50mmol). The reaction mixture is stirred for 15 minutes and then is
treated with 3-amino-5-chloro-6-methanesulfinyl-4-methyl-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide (0.345g, l.OOmmol). The reaction mixture is heated at
75°C in a sealed tube for 1.5 hours, cooled to room temperature, and then quenched by
the addition of water (10ml). The reaction mixture is transferred to an erlenmeyer flask
and diluted to 75ml with water. The mixture is cooled to 0°C and stirred. During this
time, a white precipitate is formed. This solid is collected by filtration and slurried in a
2:1 EtOAc: hexane mixture. This slurry is stirred for 10 minutes, filtered, and the
collected solid is placed in the vacuum oven to dry (60°C for 16 hours). This affords the
title compound as a white solid (0.195g, 51%). Mass (m/z): 384.1 (M++l), 382.1 (M+-l).
Example 23
3-Amino-5-chloro-4-methyl-6-[(2-pyrrolidin-l-yl-ethylcarbamoyl)-methoxy]-thieno[2,3-
b]pyridine-2-carboxylic acid cyclopropylamide
To a solution of (3-amino-5-chtoro-2-cyclopropylcarbamoyl-4-methyl-thieno[2,3-
b]pyridin-6-yloxy)-acetic acid (0.300g, O.843mmol) in a 1:1 mixture of THF:DMF (2.0ml
each) are adejed 1-hydroxybenzotriazole hydrate (0.148g, 1.096mmol), N,N-
diisopropylethylamine (0.163g, 1.265mmol), l-[3-(dimethylamino)propyi]-3-
ethylcarbodiimide hydrochloride (0.242g, 1.265mmol), and l-(2-aminoethyl)pyrrolidine
(0.289g, 2.529mmol). The resulting solution is stirred at room temperature for 16 hours.
The reaction is quenched by the addition of water (25ml). A yellow precipitate formed.
The mixture is cooled to 0°C and stirred for 10 minutes, then filtered. The collected solid
is dried in the vacuum oven for 3 hours at 60CC, This affords the title compound as a
yellow solid (0.069g, 18%). Mass (m/z): 452.2 (Mf+1), 450.2 (Ivf-1).
Example 24
3-Amino-5-chloro-6-[(4-fluoro-benzylcarbamoyl)-methoxy]-4-methyl-r.hieno[2,3-
b]pyridine-2-earboxylic acid cyclopropylamide
To a solution of (3-amino-5-ehloro-2-cyclopropylcarbamoyl-4-methyl-thieno[2,3-
b]pyridin-6-yloxy)-acetic acid (0,300g, 0.843mmol) in a 1:1 mixture of THF:DMF (2.0ml
each) are added 1 -hydroxybenzotriazole hydrate (0.148g, 1.096mmol), N,N-
diisopropylethylamine (0.163g, 1.265mmol), l-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (0.242g, 1.265mmol), and 4-fluorobenzylamine (0.317g,
2.529mmol). The resulting solution is stirred at room temperature for 16 hours. The
reaction is quenched by the addition of water (25ml). A yellow precipitate is formed.
The mixture is cooled to 0°C and stirred for 10 minutes, then filtered. The collected solid
is slurried in 10: 1 hexane:EtOAc and stirred for 10 minutes. The solid is collected by
filtration and dried in the vacuum oven at 50°C for 1 hour. This affords the title
compound as a pale yellow solid (0.l43g, 37%). Mass (m/z): 463.1 (M++l), 461.1 (M+-
1).
Example 25
3-Amino-5-chloro-4-methyl-6-[(2-morpholin-4-yl-ethylcarbamoyl)-methoxy]-thieno[2,3-
b]pyridine-2-carboxylic acid cyclopropylamide
To a solution of (3-amino-5-chloro-2-cyclopropylcarbamoyl-4-methy!-thieno[2,3-
b]pyridin-6-yloxy)-acetic acid (OJOOg, 0.843mmol) in a 1:1 mixture of THF:DMF (2.0ml
each) are added 1-hydroxybenzotriazole hydrate (0.148g, 1.096mmol), N,N-
diisopropylethylamine (0.163g, 1.265mmol), l-[3-(dimethylamino)propyl]-3-
ethylcarbodiimide hydrochloride (0.242g, 1.265mmol), and 4-(2-aminoethyl)morpholine
(0.329g, 2.529mmol). The resulting solution is stirred at room temperature for 16 hours.
The reaction is quenched by the addition of water (25ml), If no precipitate forms, the
mixture may be acidified to pH2 with IN HC1 and extracted with ElOAc (25ml). The
organic layer is discarded and the aqueous layer is made basic (pH12) with 5N NaOH and
extracted with EtOAc(40ml). The organic layer is dried (anhydrous magnesium sulfate),
filtered, and concentrated to give a yellow solid. The solid is slurried in 3:1
hexane:EtOAc (10ml), then filtered. The collected solid is dried in the vacuum oven at
50°C for 72 hours. This affords the title compound as a yellow solid (0.053g, 13%). Mass
(m/z): 468.2 (M++l), 466.2 (MM),
Example 26
3-amino-5-chloro-4-methyl-6-{2-[(pyridine-4-earbonyl)-amino]-ethoxy}-thieno[2,3-
b]pyridine-2-carboxylic acid cyclopropylamide
To a solution of N-(2-hydroxyethyl)-isonicotinamide (0,83 lg, 5.00mmol) in THF
(2.5ml) at room temperature is added dropwise a solution of lithium bis(trimethylsilyl)
amide (1.0M in hexanes)(2.50ml, 2.50mmol). The reaction mixture is stirred for 15
minutes and then is treated with 3-amino-5-chloro-6-inethanesulfinyl-4-methyl-
thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (0.345g, l.OOmmol). The
reaction mixture is heated at 80°C in a sealed tube for 2 hours, cooled to room
temperature, and then quenched by the addition of water (15ml). The mixture is diluted
to 100ml with water and then cooled to 0°C and stirred. A yellow precipitate is formed.
This solid is collected by filtration, slurried in EtOAc, and filtered again to give a yellow
solid which is placed in the vacuum oven to dry (50CC for 3 hours). This affords the title
compound as a yellow solid (0.046g, 10%). Mass (m/z): 446.3 (M++l), 444.2 (M+-l).
Example 27
3-Amino-6-benzyloxy-5-chloro-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid
cycloprcpylamide
To a solution of benzyl alcohol (0.541g, 5.00mraol) in THF (2.5ml) at room
temperature is added dropwise a solution of lithium bis(trimethylsilyl')amide (1.0M ;n
hexanes)(2.50ml, 2.50mmol), The reaction mixture is stirred for 15 minutes and then is
treated with 3-amino-5-chloro-6-methanesulfinyl-4-methyl-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide (0.345g, l.OOmmol). The reaction mixture is heated at
80°C in a sealed tube for 2 hours, cooled to room temperature, and then quenched by the
addition of water (15ml). The mixture is diluted to 100ml with water and then cooled to
0CC and stirred. A yellow precipitate is formed. This solid is collected by filtration and
purified by flash chromatography (1.5: 1 hexane: EtOAc) to give the title compound as a
pale yellow solid (0.130g, 33%). Mass (m/z): 388.2 (M+-l), 386.2 (M+-l).
Example 28
3-Amino-5-chloro-4-methyl-6-(2-morpholin-4-yl-ethoxy)-thieno[2,3-bjpyridine-2-
carboxylic acid cyclopropylamide hydrochloride
A mixture of 3-amino-5-chloro-4-methyl-6-(2-morpholin-4-yl-ethoxy)-thieno[2,3-
b]pyridine-2-carboxylic acid cyclopropylamide (0.800g, 1,95mmol) and MeOH (50ml) is
heated to 50°C. The mixture is treated with THF (5ml) and DMF (5ml). While still at
50°C, the mixture is acidified to pHl with concentrated HC1. A homogeneous solution is
formed. The solution is allowed to cool slowly to room temperature. During this time, a
white precipitate is formed. This solid is collected by Filtration and dried in the vacuum
oven for 72 hours at 50°C. This affords the title compound as a white solid (0.562g,
65%). Mass (m/z): 411.1 (M++1)-HC1, 445.1 (M+-1).
Example 29
3-Amino-5-chloro-6-ethoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid
cyclopropylamide
Sodium hydride (0.037g, 0.925mmol) is suspended in anhydrous 1,2-dimethoxy-
ethane (5.0ml) under nitrogen. The suspension is cooled to 0°C. To this cold suspension
is added via cannula a solution of 3-amino-5-chloro-6-hydroxy-4-methyl-thieno[2,3-
b]pyridine-2-carboxylic acid cyclopropylamide (0.300g, l.Olmmol) in anhydrous DMF
(5.0ml). The ice bath is removed, and the reaction is warmed to room temperature. After
the reaction is stirred 25 minutes at room temperature, lithium bromide (0.175g,
2.02mmol) is added as a solid, and the reaction is stirred for an additional two hours.
Finally, ethyl iodide is added (0.24ml, 0.47g, 3.00mmol), and the reaction is stirred for 36
hours at room temperature. At the completion of the reaction time, the mixture is
quenched with water. The solid is collected by filtration, then purified by flash
chromatography (hexanes:EtOAc grathent) to give the title compound as a white solid
(24.3mg, 8% yield). Mass (m/z): 326.0 (M1"), 324.0 (M-).
Alternatively, Example 33 may be prepared following the procedures outlined for
the preparation of Example 29, substituting EtOH for benzyl alcohol.
Example 30
(3-Amino-5-chloro-2-cyclopropylcarbamoyl-4-methyl-thieno[2,3-b]pyridin-6-yloxy)-
acetic acid (imidazol-l-yl)-amide
To a solution of (3-amino-5-chloro-2-cyclopropylcarbamoyl-4-methyl-thieno[2,3-
b]pyridin-6-yloxy)-acetic acid (0.350g, 0.98mmol) in DMF (4ml) at room temperature is
added 1,1 '-carbonyldiimidazole (0.239g, 1.48mmol). The resulting solution is heated and
stirred at 40°C for 20 minutes. During this time, a cream colored precipitate is formed.
The mixture is cooled to 10°C, and neat 2-aminopyridine (0.231g, 2.46mmol) is added.
The mixture is stirred at room temperature for 30 minutes, then quenched by adding
saturated sodium bicarbonate solution (10ml). A thick, white precipitate is formed. The
mixture is diluted with water (25ml), stirred for 10 minutes, then filtered. The collected
solid is dried in the vacuum oven for 16 hours. This affords the title compound as a white
solid (0.228g, 58%). Mass (m/z): 404.0 (MM).
The compounds of the present invention can be administered alone or in the form
of a pharmaceutical composition, that is, combined with pharmaceutically acceptable
carriers, or excipients, the proportion and nature of which are determined by the solubility
and chemical properties of the compound selected, the chosen route of administration,
and standard pharmaceutical practice. The compounds of the present invention, while
effective themselves, may be formulated and administered in the form of their
pharmaceutically acceptable salts, for purposes of stability, convenience of
crystallization, increased solubility, and the like.
Thus, the present invention provides pharmaceutical compositions comprising a
compound of the Formula 1 and a pharmaceutically acceptable diluent.
The compounds of Formula I can be administered by a variety of routes. In
effecting treatment of a patient afflicted with disorders described herein, a compound of
Formula I can be administered in any form or mode that makes the compound
bioavailable in an effective amount, including oral and parenteral routes. For example,
compounds of Formula I can be administered orally, by inhalation, or by the
subcutaneous, intramuscular, intravenous, transdermal, intranasal, rectal, occular, topical,
sublingual, buccal, or other routes. Oral administration is generally preferred for
treatment of the neurological and psychiatric disorders described herein.
One skilled in the art of preparing formulations can readily select the proper form
and mode of administration depending upon the particular characteristics of the
compound selected, the disorder or condition to be treated, the stage of the disorder or
condition, and other relevant circumstances. (Remington's Pharmaceutical Sciences, 18th
Edition, Mack Publishing Co. (1990)).
The pharmaceutical compositions are prepared in a manner well known in the
pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material
that can serve as a vehicle or medium for the active ingrethent. Suitable carriers or
excipients are well known in the art. The pharmaceutical composition may be adapted for
oral, inhalation, parenteral, or topical use and may be administered to the patient in the
form of tablets, capsules, aerosols, inhalants, suppositories, solutions, suspensions, or the
like.
The compounds of the present invention may be administered orally, for example,
with an inert diluent or capsules or compressed into tablets. For the purpose of oral
therapeutic administration, the compounds may be incorporated with excipients and used
in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing
gums and the like. These preparations should contain at least 4% of the compound of the
present invention, the active ingrethent, but may be varied depending upon the particular
form and may conveniently be between 4% to about 70% of the weight of the unit. The
amount of the compound present in compositions is such that a suitable dosage will be
obtained. Preferred compositions and preparations according to the present invention
may be determined by a person skilled in the art.
The tablets, pills, capsules, troches, and the like may also contain one or more of
the following adjuvants: binders such as povidone, hydroxypropyl cellulose,
microcrystalline cellulose, gum tragacanth or gelatin; excipients such as dicalcium
phosphate, starch, or lactose; disintegrating agents such as alginic acid, Primogel, corn
starch and the like; lubricants such as talc, magnesium stearate or Sterotex; glidants such
as colloidal silicon dioxide; and sweetening agents, such as sucrose, aspartame, or
saccharin, or a flavoring agent, such as peppermint, methyl salicylate or orange flavoring,
may be added. When the dosage unit form is a capsule, it may contain, in addition to
materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
Other dosage unit forms may contain other various materials that modify the physical
form of the dosage unit, for example, coalings. Thus, tablets or pills may be coated with
sugar, shellac, or other coating agents. A syrup may contain, in addition to the present
compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings
and flavors, Materials used in preparing these various compositions should be
pharmaceutically pure and non-toxic in the amounts used.
For the purpose of parenteral therapeutic administration, the compounds of the
present invention may be incorporated into a solution or suspension. These preparations
typically contain at least 0.001% of a compound of the invention, but may be varied to be
between 0.001 and about 90% of the weight thereof. The amount of the compound of
Formula I present in such compositions is such that a suitable dosage will be obtained.
The solutions or suspensions may also include one or more of the following adjuvants:
sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene
glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such
as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium
bisulfite; chelating agents, such as ethylene diaminetetraacetic acid, buffers, such as
acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium
chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic. Preferred compositions and
preparations are able to be determined by one skilled in the art.
The compounds of the present invention may also be administered topicaily, and
when done so, the carrier may suitably comprise a solution, ointment, or gel base. The
base, for example, may comprise one or more of the following: petrolatum, lanolin,
polyethylene glycols, bees wax, mineral oil, diluents such as water and alcohol, and
emulsifiers, and stabilizers. Topical formulations may contain a concentration of a
compound of Formula I or its pharmaceutical salt from about 0.1 to about 10% w/v
(weight per unit volume).
The compounds of Formula 1 are allosteric potentiators of the M4 subtype of
muscarinic receptors. Furthermore, the compounds of Formula 1 selectively potentiate
M4 receptors relative to other muscarinic receptors. The activity of the compounds of the
present invention may be determined by the methods below.
Calcium mobilization in whole cells stably expressing human muscarinic receptors
A. Stable Cell Lines
Standard molecular cloning techniques may be used to generate stable cell lines
expressing the human muscarinic M1-M5 receptors. M,, M3 an M5 receptors arc
expressed in Chinese hamster ovary (CHO) cell lines, whereas, M2 an M4 are expressed in
AVI2 Gai5 cell lines. The cDNA encoding these muscarinic receptors correspond to the
published sequence in the NCBI nucleotide database of accession numbers: AF498915,
AF498916, AF498917, AF498918 and AF498919, forM,-M5 respectively.
B. Methods
Using a calcium-sensitive fluorescent dye, agonist or potentiation activity of a
given compound can be detected in a single assay using a Fluorescence Light Imaging
Plate Reader (FLIPR) instrument. Cells are plated in Poly-D-Lysine coated black
plates/clear bottom (Becton Dickinsons) at 40,000 cells per ml (lOOul/well) in growth
media 24 hours prior to running the assay. The medium is removed before the addition of
50u,l fluorescence dye solution (HBSS containing 20mM Hepes, 10u.M Fluo-3-AM,
0.05% pluronic acid F127; supplemented with 2.5mM probenecid for CHO cell assays).
The cells are incubated with the dye for 75 minutes before replacing with assay buffer
(20mM Hepes in Hanks balanced salt solution (Gibco); supplemented with 2.5mM
probenecid for CHO cells). The plate is transferred to the FLIPR machine (Molecular
Devices) for fluorescence recordings. The cells are periodically excited by 488nm light,
and the emitted fluorescent light passed through a 510-570nm filter and then detected by
a cooled CCD camera. Automated multiple compound additions are timed by computer
program. Cells are pre-incubated with increasing concentration of compounds. After 2
minutes, a range of acetylcholine concentrations is added to each concentration of
compound. If the compound is an allosteric enhancer, a compound concentration-
dependent potentiation of acetylcholine response would be detectable. The effectiveness
of potentiator compounds can be ranked by their affinity and cooperativity.
An alternative method may be used to provide an estimate of the affinity of
compounds of the present invention, and to rank order the compounds based on this
estimated affinity. In this method, a single concentration of carbachol that is
approximately 10% of a saturating concentration is added to all wells, and increasing
concentrations of the test compounds of the present invention are added. An estimated
affinity value is derived by calculation of the EC50 for potentiation of the 10% carbachol
response. This method may be used to rank order the compounds described herein as
Examples.
As confirmed with the compound of Example 1, acetylcholine and carbachol (both
non-selective full muscarinic receptor agonists) are potentiated in an equivalent fashion in
the presence of an allosteric modulator,
C. Data Analysis and Results
Allosteric parameters may be estimated using the equations by Lazareno et al.,
Mo!. Pharmacol. (1995) 48: 362-378. The effect of increasing concentrations of the
compound of Example 1 on cellular ACh concentration-response curves in recombinant
cell lines (AV12 Ga15 hM2 or hM4 and CHO hMi, I1M3 or hMs) may be tested using
FLIPR. Data are collected in duplicates from at least three independent experiments. No
significant allosteric effect is observed in CHO cells stably expressing hMi, hM3 or hM5
receptors. The cooperativity factor and affinity of the compound of Example 1 for hM4
receptors is estimated to be 34.5 ± 3.5 and 200 ± 42 nM, respectively. A modest
allosteric effect on hM2 receptors is also observed but is too modest to be accurately
estimated.
Using the FLIPR assay to potentiate 10% carbachol, as described above for rank
ordering of the compounds of the present invention, each of the compounds described
herein as EXAMPLES has an estimated affinity for hM^ receptors of < 500 11M.
Neurotransmitter release assay
A. Methods
Two male Lister Hooded rats are killed by carbon dioxide asphyxiation and
cervical dislocation. The brains are rapidly removed, and the striatum dissected out and
cross-chopped three times at 150pm. The slices are suspended in 12ml of HEPES buffer
(128mM NaCl, 2.4mM KCI, 3.2mM CaCl2, 1.2mM KH2PO4, 1.2 mM MgSO47H20,
25mM HEPES, lOmM Glucose, pH 7.5). The slices are washed twice, with resuspending
in fresh buffer each time, then incubated at 37°C for 30 minutes with [3H]-choline
chloride (250nM). After 30 minutes, a further four washes are carried out, and 100|il of
slices are placed in each well of a 96 well filter plate (Millipore MABCN 96-well
multiscreen plate). The bathing solution is removed by vacuum filtration (Millipore
Univac manifold system), then a further 70p.l of HEPES buffer (+/- compound) is added
to each well, and the plate is returned to the incubator for 5 minutes. After a 5-minute
incubation, the buffer is removed by vacuum filtration into a collecting plate (Wallac 96
well flexible sample plates). Stimulating solution (70nl/well: 20mM potassium 1-/-
compound) is then added, and the plate returned to the incubator for a further 5-minutes.
The stimulating buffer is then removed by vacuum filtration into a second collecting
plate. At the end of the experiment, the amount of tissue in each well is estimated by
placing the plates in a freezer for one hour, punching out the filter discs, and adding
Soluene® (to digest the slices) and leaving for a further hour. The radioactivity in the
digested tissue is measured using liquid scintillation counting. Neurotransmitter release is
calculated as a fraction of total radioactivity present in the well.
B. Results
The potentiation effect of compounds in native tissues is tested by its ability to
potentiate auto-inhibition of acetylcholine release in striatal slices, as induced by 20mM
potassium stimulation. This is considered to be an M4-mediated process via pre-synaptic
auto-regulation in the striatum. Zhang,W. et al, J.Neurosci. (2002)22: 1709-1717.
Using the methods above, a representative compound of the present invention (Example
1) exhibits concentration-dependent potentiation of auto-inhibition with an 1C5[) of 1.5
uM.
Several preclinical laboratory animal models have been described for a number of
the disorders associated with muscarinic receptors. For instance, inhibition of the
conditioned avoidance response (CAR) by neuroleptic and atypical antipsychotics is one
of the most stuthed pharmacological models of psychosis. To date, all clinically effective
antipsychotics have been demonstrated to selectively suppress CAR (cf. Wadenberg &
Hicks, 1999. Neuroscience & Biobehavioral Reviews, 23: 851-8).
Conditioned Avoidance Response
A. Methods
Male Fisher-344 rats (N = 5-8) are trained in an avoidance paradigm in which the
rat must make a shuttle response to avoid or escape a footshock. The apparatus is a
Coulbourn Instruments shuttle operant chamber. Each session equals a total of 50 trials,
presented on a 30 second inter-trial interval, and each trial begins with simultaneous
illumination of a houselight and rising of a guillotine door. The rat is given 10 seconds to
cross over to the other side (an avoidance response) before a 1 mA footshock is initiated.
The shock remains on until the rat crosses over to the other side (an escape response) or
10 seconds has elapsed (an escape failure). Rats are well trained on this task, with
baseline avoidance performance > 95 %. The number of avoidance, escape and escape
failure responses during each session is recorded and used for analysis.
Groups of rats are dosed with (1) vehicle (10% acacia plus sterile water), (2) a
sub-efficacious dose of the muscarinic agonist oxotremorine sesquifumarate (Oxo alone),
or (3) Oxo in the presence of increasing doses of the test compound (10 mg/kg to 60
mg/kg), followed by (4) a retest of Oxo alone. Each test compound is administered orally
2 hours prior to testing. Oxo (0.03 mg/kg) is administered subcutaneously 30 minutes
prior to testing. Data are analyzed via a one-way (within-group design) Analysis of
Variance (ANOVA). In cases of a significant ANOVA (p < 0.05), post-hoc comparisons
may be made in which compound doses are compared back to the Oxo alone group
(paired t-test).
The conditioned avoidance assay is highly predictive of antipsychotic efficacy in
the clinic. Representative muscarinic M4 receptor potentiators exhibit an antipsychotic-
like profile in the conditioned avoidance responding paradigm. Although these M4
potentiators are inactive when tested alone (data not shown), these compounds potentiate
the efficacy of an inactive dose of the muscarinic agonist oxotremorine.
The results of calcium mobilization and neurotransmitter release stuthes
demonstrate the ability of compounds of the present invention to act as potentiators of M4
muscarinic receptors. It is recognized that the compounds of the present invention would
be expected to potentiate the effects of M4 receptor activation. Thus, the compounds of
the present invention are expected tc be useful in the treatment of various disorders
associated with muscarinic receptors, as described to be treated herein, and other
disorders that can be treated by such allosteric potentiators, as are appreciated by those
skilled in the art.
The disorders associated with M4 muscarinic receptors are treated by
administering an effective amount of a compound or pharmaceutical composition of
Formula I. An effective amount can be readily determined by the attending diagnostician,
as one skilled in the art, by the use of conventional techniques and by observing results
obtained under analogous circumstances. In determining an effective amount, the dose of
a compound of Formula I, a number of factors are considered by the attending
diagnostician, including, but not limited to: the compound of Formula I to be
administered; the species of mammal - its size, age, and general health; the specific
disorder involved; the degree of involvement or the severity of the disorder; the response
of the individual patient; the mode of administration; the bioavailability characteristics of
the preparation administered; the dose regimen selected; the use of other concomitant
medication; and other relevant circumstances.
An effective amount of a compound of Formula 1 is expected to vary from about
0.001 milligram per kilogram of body weight per day (mg/kg/day) to about 100
mg/kg/day. Preferred amounts may be readily determined by one skilled in the art.
We Claim:
1. Novel thienopyridine modulators of the M4 Muscarinic Receptor of Formula I:
wherein:
m is 1 or 2
n is 0 or 1
X is a bond
R1 is hydrogen or morpholino
or a pharmaceutically acceptable salt thereof.
2. The compound as claimed in claim 1, wherein the compound is selected from
i) 3-Amino 5-chloro 6 methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid
cyclopropylamide or pharmaceutically acceptable salt thereof;
ii) 3-Amino-5-chloro-4-methyl-6-(2-morpholin-4-yl-ethoxy)-thieno[2,3-b]pyridine-2-
carboxylic acid cyclopropylamide or pharmaceutically acceptable salt thereof;
3. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically
salt thereof in combination with pharmaceutically acceptable carrier, excipients or diluents,
4. A compound and a pharmaceutical composition to treat the conditions associated with M4
muscarinic receptor activity, substantially as herein described and as illustrated in the
foregoing examples.
The present invention relates to selective allosteric potentiators of the Formula (I):
or pharmaceutically acceptable salts thereof, for the treatment of disorders associated with M4
muscarinic receptors.