The present invention relates to compounds that are potentiators of the
hMrgX1receptor, to pharmaceutical compositions comprising the compounds, to methods
of using the compounds to treat pain, and to intermediates and processes useful in the
synthesis of the compounds.
It is estimated that about 20% of adults in the United States alone suffer from
10 chronic pain. Chronic pain is one of the most common reasons adults seek medical care
and is linked to restrictions in mobility and daily activities. Unfortunately, chronic pain is
often refractory to current therapies and many analgesics are associated with doselimiting
adverse events or serious risk of addiction and abuse which can be substantial
barriers to their use in treating chronic pain. Thus, there is an unmet need for new
15 chronic pain therapies, particularly treatments that have such adverse effects reduced or
effectively eliminated.
United States Patent No. 6,326,368 B 1 discloses certain 2-aryloxy- and 2-
arylthiosubstituted pyrimidines and triazines and derivatives thereof as corticotropin
releasing factor (CRF) receptor antagonists useful in treating various disorders, such as
20 depression, anxiety, drug addiction, and inflammatory disorders. United States Patent
No. 5,100,459 discloses certain substituted sulfonylureas and intermediates thereof W.
Wangdong, et. al., ChemMedChem, vol 10(1), 57-61 (2015) disclose 2-
(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382, as a potent and
selective positive allosteric modulator ofMrgXl.
25 There is a need for alternate treatments of pain including chronic pain. In
addition, there is a need for compounds that are potentiators of the hMrgX1 receptor.
5
10
15
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Accordingly, in one embodiment, the present invention provides a compound of
Formula 1:
wherein R1 is hydrogen or methyl; and
R2 is:
Formula I
d
~JlAF ,
F
~
~~ , or
~
~~N
or a pharmaceutically acceptable salt thereof.
In an embodiment, R 1 is hydrogen.
In an embodiment, R 1 is methyl.
In an embodiment, R2 is:
In an embodiment, R2 is:
F 56
In an embodiment, R2 is:
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In a particular embodiment, the compound is:
or a pharmaceutically acceptable salt thereof.
In a particular embodiment, the compound is:
5
or a pharmaceutically acceptable salt thereof.
In a particular embodiment, the compound is:
or a pharmaceutically acceptable salt thereof.
10 In a particular embodiment, the compound is:
or a pharmaceutically acceptable salt thereof.
In a particular embodiment, the compound is:
15 or a pharmaceutically acceptable salt thereof.
5
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In a particular embodiment, the compound is:
or a pharmaceutically acceptable salt thereof
In an embodiment, the present invention also provides a method of treating pain in
a patient in need of such treatment, comprising administering to the patient an effective
amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof In an
embodiment, the present invention further provides a method of treating chronic pain in a
patient in need of such treatment, comprising administering to the patient an effective
10 amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof In an
embodiment, the present invention further provides a method of treating chronic lower
back pain in a patient in need of such treatment, comprising administering to the patient
an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt
thereof In an embodiment, the present invention further provides a method of treating
15 diabetic peripheral neuropathic pain in a patient in need of such treatment, comprising
administering to the patient an effective amount of a compound ofFormula I, or a
pharmaceutically acceptable salt thereof In an embodiment, the present invention further
provides a method of treating osteoarthritis pain in a patient in need of such treatment,
comprising administering to the patient an effective amount of a compound of Formula I,
20 or a pharmaceutically acceptable salt thereof
In an embodiment, the present invention further provides a compound of Formula
I, or a pharmaceutically acceptable salt thereof for use in therapy. In an embodiment, the
present invention provides a compound of Formula I, or a pharmaceutically acceptable
salt thereof for use in treating pain. In an embodiment, the present invention provides a
25 compound of Formula I, or a pharmaceutically acceptable salt thereof, for use in treating
chronic pain.
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In an embodiment, the present invention also provides the use of a compound of
Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for treating pain. In an embodiment, the present invention provides the use
of a compound of Formula I, or a pharmaceutically acceptable salt thereof, for the
5 manufacture of a medicament for treating chronic pain.
In an embodiment, the present invention further provides a pharmaceutical
composition, comprising a compound of Formula I, or a pharmaceutically acceptable salt
thereof, with one or more pharmaceutically acceptable carriers, diluents, or excipients. In
an embodiment, the present invention further provides a process for preparing a
10 pharmaceutical composition, comprising admixing a compound ofFormula I, or a
pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable
carriers, diluents, or excipients. In an embodiment, the present invention also
encompasses novel intermediates and processes for the synthesis of compounds of
Formula I.
15 As used herein, the terms "treating", "treatment'', or "to treat" includes
restraining, slowing, stopping, or reversing the progression or severity of an existing
symptom or disorder.
As used herein, the term "patient" refers to a mammal, in particular a human.
As used herein, the term "effective amount" refers to the amount or dose of
20 compound of the invention, or a pharmaceutically acceptable salt thereof which, upon
single or multiple dose administration to the patient, provides the desired effect in the
patient under diagnosis or treatment.
An effective amount can be determined by one skilled in the art by the use of
known techniques and by observing results obtained under analogous circumstances. In
25 determining the effective amount for a patient, a number of factors are considered by the
attending diagnostician, including, but not limited to: the species of patient; its size, age,
and general health; the specific disease or disorder involved; the degree of or involvement
or the severity of the disease or disorder; the response of the individual patient; the
particular compound administered; the mode of administration; the bioavailability
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characteristics of the preparation administered; the dose regimen selected; the use of
concomitant medication; and other relevant circumstances.
The compounds of the present invention are formulated as pharmaceutical
compositions administered by any route which makes the compound bioavailable. Most
5 preferably, such compositions are for oral administration. Such pharmaceutical
compositions and processes for preparing same are well known in the art (See, e.g.,
Remington: The Science and Practice of Pharmacy, L.V. Allen, Editor, 22nd Edition,
Pharmaceutical Press, 2012).
Certain intermediates described in the following preparations may contain one or
10 more nitrogen protecting groups. It is understood that protecting groups may be varied as
appreciated by one of skill in the art depending on the particular reaction conditions and
the particular transformations to be performed. The protection and deprotection
conditions are well known to the skilled artisan and are described in the literature (See for
example "Greene's Protective Groups in Organic Synthesis", Fourth Edition, by Peter
15 G.M. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2007).
A pharmaceutically acceptable salt of a compound of the invention can be formed,
for example, by reaction of an appropriate free base of a compound of the invention, an
appropriate pharmaceutically acceptable acid in a suitable solvent such as diethyl ether
under standard conditions well known in the art. Additionally, the formation of such
20 pharmaceutically acceptable salts can occur simultaneously upon deprotection of a
nitrogen protecting group. See, for example, Gould, P.L., "Salt selection for basic drugs,"
International Journal of Pharmaceutics, 33: 201-217 (1986); Bastin, R.J., et al. "Salt
Selection and Optimization Procedures for Pharmaceutical New Chemical Entities,"
Organic Process Research and Development, 4: 427-435 (2000); and Berge, S.M., et al.,
25 "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977).
Certain abbreviations are defined as follows: "ACN" refers to acetonitrile;
"BAM8-22" refers to bovine adrenal medulla peptide 8-22; "Cat. #" refers to catalog
number; "CRC" refers to concentration-response curve; "DMEM" refers to Dulbecco' s
modified eagle media; "DMSO" refers to dimethyl sulfoxide; "DPBS" refers to
30 Dulbecco' s phosphate-buffered saline; "ECso" refers to the effective concentration of an
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agent that gives a half-maximal response between baseline and maximum after a specified
exposure time; "EDTA" refers to ethylenediaminetetraacetic acid; "ESMS" refers to
Electrospray Mass Spectrometry; "FBS" refers to fetal bovine serum; "g" refers to gram
or grams; "h" refers to hour or hours; "HEC" refers to hydroxyethylcellulose; "HEK293"
5 refers to human embryonic kidney 293 cell or cells; "HEPES" refers to ( 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid); "hMrgX1" refers to human MrgX1
receptor; "HTRF" refers to homogeneous time resolved fluorescence; "IP1" refers to
inositol monophosphate; "Kp,uu" refers to unbound brain-to-plasma partition coefficient;
"LC-ESMS" refers to refers to Liquid Chromatography Electrospray Mass Spectrometry;
10 "min" refers to minute or minutes; "mL" refers to milliliter or milliliters; "Me" refers to
methyl; "mol" refers to mole or moles; "mmol" refers to millimole or millimoles; "nm"
refers to nanometer or nanometers; "nmol" refers to nanomoles; "m/z" refers to mass-tocharge
ration for mass spectroscopy; "n," when in the context of biological data, refers to
the number of runs or number of times tested; "PBS" refers to phosphate-buffered saline;
15 "rpm" refers to revolutions per minute or minutes; "SD" refers to standard deviation;
"SEM" refers to standard error of the mean; "U/mL" refers to units per milliliter.
General Chemistry
The compounds of the present invention, or salts thereof, may be prepared by a
20 variety of procedures known to one of ordinary skill in the art, some of which are
illustrated in the schemes, preparations, and examples below. One of ordinary skill in the
art recognizes that the specific synthetic steps for each of the routes described may be
combined in different ways, or in conjunction with steps from different schemes, to
prepare compounds of the invention, or salts thereof The products of each step in the
25 schemes below can be recovered by conventional methods well known in the art,
including extraction, evaporation, precipitation, chromatography, filtration, trituration,
and crystallization. In the schemes below, all substituents unless otherwise indicated, are
as previously defined. The reagents and starting materials are readily available to one of
ordinary skill in the art. The following schemes, preparations, examples, and assays
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further illustrate the invention, but should not be construed to limit the scope of the
invention in any way.
Scheme 1
+
LG =e.g., Cl, Br, I, triflate, mesylate, tosylate Formula I
5 Scheme 1 depicts a general preparation of compounds of Formula I (R1 = H or
CH3; R2 = 2,6-difluorophenyl, 2,4,6-trifluorophenyl, or 4-cyano-2,6-difluorophenyl) via
nucleophilic aromatic substitution, as is well known to a person of ordinary skill in the
art. Additionally, compounds of Formula I may be prepared via transition-metal (e.g.,
copper-, nickel-, or palladium-mediated) cross-coupling or Ullmann-type reactions as is
10 well described in the art.
The 2-amino-6-trifluoromethyl-5-substituted pyrimidine starting material with an
appropriate leaving group (LG) at the 4-position (e.g., LG = Cl, Br, I, triflate, mesylate,
tosylate) may be purchased commercially. Alternatively, a person of ordinary skill in the
art will recognize that the appropriate starting material may be prepared under a variety of
15 techniques well documented in the art, such as dehydrative cyclization of guanidine with
an appropriate 5-substituted 4,4,4-trifluoro-2-methyl-3-oxo-butanoic acid ester (R1 = H,
CH3) under basic conditions. Conversion of the subsequent cyclized 2-amino-6-
trifluoromethyl-4-hydroxy-5-substituted pyrimidine to an appropriate LG at the 4-position
is well recognized to a person of ordinary skill in the art.
20
Preparations and Examples
The following Preparations and Examples further illustrate the invention and
represent typical synthesis of the compound of the invention. The reagents and starting
materials are readily available or may be readily synthesized by one of ordinary skill in
25 the art. It should be understood that the Preparations and Examples are set forth by way
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of illustration and not limitation, and that various modifications may be made by one of
ordinary skill in the art.
LC-ES/MS is performed on an AGILENT® HP1100 liquid chromatography
system. Electrospray mass spectrometry measurements are performed on a Mass
5 Selective Detector quadrupole mass spectrometer interfaced to the HP1100 HPLC. LCMS
conditions (low pH): column: PHENOMENEX® GEMINI® NX C18 2.1 x 50 mm
3.0 11-m; gradient: 5-100% Bin 3 min, then 100% B for 0.75 min column temperature: 50
oc +/-10 oc; flow rate: 1.2 mL/min; Solvent A: deionized water with 0.1% HCOOH;
Solvent B: ACN with 0.1% formic acid; wavelength 214 nm. Alternate LC-MS
10 conditions (high pH): column: XTERRA® MS C18 columns 2.1x50 mm, 3.5 11-m;
gradient: 5% of solvent A for 0.25 min, gradient from 5% to 100% of solvent Bin 3 min
and 1 00% of sol vent B for 0. 5 min or 1 0% to 1 00% of sol vent B in 3 min and at 1 00% of
solvent B for 0.75 min; column temperature: 50 oc +/-10 oc; flow rate: 1.2 mL/min;
Solvent A: 10 mM NH4HC03 pH 9; Solvent B: ACN; wavelength: 214 nm.
15
Preparation 1
4-( 4-Bromo-2, 6-difl uoro-phenoxy )-6-( trifl uoromethy 1 )pyrimi din-2-amine
F
F3cyyo~
NyN FA)lBr
NH2
Combine 2-amino-4-chloro-6-(trifluoromethyl)pyrimidine (500 mg, 2.4 mmol)
20 and 4-bromo-2,6-difluorophenol (616 mg, 2.9 mmol) to a microwave vial and add ACN
(10 mL). Add potassium carbonate (665 mg, 4.8 mmol), seal the vial, and heat at 160 °C
for 1 h in a microwave reactor. Cool the reaction mixture, dilute with water, and extract
three times with ethyl acetate. Combine the organic extracts and dry over sodium sulfate.
Filter and evaporate the resulting filtrate under reduced pressure. Purify the resulting
25 residue by flash chromatography over silica gel, using a gradient of 5-100% ethyl acetate
in hexanes, to afford the title compound (789 mg, 89% yield), after solvent evaporation of
the desired chromatographic fractions. ESMS (m/z, 79Br/81Br): 370/372 [M+H].
5
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Preparation 2
2-Amino-5-methyl-6-(trifluoromethyl)pyrimidin-4-ol
CH3
F3cyYoH
N'fN
NH2
Add a 25% solution of sodium methoxide in methanol (5.5 mL, 24 mmol) to a
solution of ethyl 4,4,4-trifluoro-2-methyl-3-oxo-butanoate (4 g, 20 mmol) and guanidine
(1.2 g, 20 mmol) in methanol (100 mL). Stir for 23 hat room temperature. Evaporate the
reaction mixture under reduced pressure. Dissolve the resulting white solid in water (20
mL) and acidify with acetic acid (2 mL). Collect the resulting product by vacuum
10 filtration, wash twice with water, and dry the resulting filter cake under vacuum to obtain
15
the title compound (2.3 g, 60% yield) as a white solid. ESMS (m/z): 194 [M+H].
Preparation 3
4-Chloro-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine
CH3
F3cyYc1
NyN
NH2
In a microwave vial, add phosphoryl chloride (5.5 mL, 59 mmol) to 2-amino-5-
methyl-6-(trifluoromethyl)pyrimidin-4-ol (2.3 g, 11.9 mmol). Heat the reaction mixture
at 110 oc for 30 min in a microwave reactor. Pour the reaction mixture onto ice, basify
with 5 M aqueous NaOH (50 mL), and extract with ethyl acetate (100 mL). Dry the
20 combined extracts over sodium sulfate, filter, and evaporate the resulting filtrate under
reduced pressure. Dissolve the resulting residue in dichloromethane and purify by flash
chromatography over silica gel, eluting with 10-25% ethyl acetate in hexanes, to obtain
the title compound (813mg, 32% yield) as a white solid, after evaporation of the desired
chromatographic fractions. ESMS (m/z, 35Cl/37Cl): 212/214 [M+H].
5
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Preparation 4
4-Chloro-5-iodo-6-(trifluoromethyl)pyrimidin-2-amine
Cl
I~N
F3CANJl.N H2
Combine 4-chloro-6-(trifluoromethyl)pyrimidin-2-amine (5 g, 25.2 mmol) in
acetic acid (300 mL) with N-iodosuccinimide (32.0 g, 138 mmol) at 0 °C. Heat and stir
the resulting mixture at 70°C overnight. Cool the reaction and quench with water (120
mL). Extract with ethyl acetate (100 mL x 2). Wash the combined organic layers with
brine (50 mL x 2), dry over Na2S04, filter, and concentrate under reduced pressure. Purify
10 the crude product by flash chromatography to afford 4.8 g (53% yield) of the title product
as a yellow solid. ES/MS m/z 324[M+Ht
15
Preparation 5
4-((2-Amino-5-iodo-6-(trifluoromethyl)pyrimidin-4-yl)oxy)-3,5-difluorobenzonitrile
Combine 4-chloro-5-iodo-6-(trifluoromethyl)pyrimidin-2-amine (2.5 g, 7.0 mmol,
90 mass%) and 3,5-difluoro-4-hydroxy-benzonitrile (1.33 g, 8.49 mmol) in DMF (35 mL)
and add potassium carbonate (2.88 g, 20.8 mmol). Heat the reaction mixture at 90 oc for
3 h. Cool the reaction mixture and quench with water (100 mL). Extract with ethyl
20 acetate (60 mL x 2) and wash the combined organic layers with brine (50 mL x 2), dry
over Na2S04, filter, and concentrate under reduced pressure. Purify the crude product
with flash chromatography to afford 2.6 g (76% yield) of the title product as a white solid.
ES/MS m/z 443 [M+Ht
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Example 1
4-(2, 6-Difl uorophenoxy )-6-( trifl uoromethy 1 )pyrimi din-2-amine
F
F3cyyo~
NyN FA)
NH2
In a microwave vial, add potassium tert-butoxide (270 mg, 2.4 mmol) to a solution
5 of 2-amino-4-chloro-6-(trifluoromethyl)pyrimidine (395 mg, 2.0 mmol) and 2,6-
difluorophenol (289 mg, 2.2 mmol) in ACN (8.0 mL). Heat the reaction mixture at 120
oc for 30 min in a microwave reactor. Filter the reaction mixture through diatomaceous
earth and evaporate the filtrate under reduced pressure. Dissolve the resulting residue in
dichloromethane containing a small amount of methanol and purify the mixture by flash
10 chromatography over silica gel, eluting with a gradient of 5-20% ethyl acetate in hexanes,
to obtain the title compound (512 mg, 88% yield) as a white crystalline solid, after
solvent evaporation of the desired chromatographic fractions. ESMS (m/z): 292 [M+H].
15
Example 2
4-(2, 4, 6-Trifl uorophenoxy )-6-( trifl uorom ethy 1 )pyrimi din-2-amine
F
F3cyyo~
NyN FAJlF
NH2
In a 500-mL round-bottom flask, add potassium carbonate (26.9 g, 194.7 mmol) to
a solution of2-amino-4-chloro-6-(trifluoromethyl)pyrimidine (19.55 g, 97 mmol) and
2,4,6-trifluorophenol (15.2 g, 97.5 mmol) in N,N-dimethylformamide (200 mL). Heat the
20 reaction mixture at 80 °C for 16 h. Quench the reaction mixture with water (500 mL) and
extract with ethyl acetate (2 x 500 mL). Wash the combined organic extracts with
saturated aqueous NaCl (2 x 800 mL), dry over sodium sulfate, filter, and concentrate the
resulting filtrate under reduced pressure. Purify the resulting residue by flash
chromatography over silica gel, eluting with a gradient of 0-37% ethyl acetate in
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petroleum ether, to obtain the title compound (15.9 g, 53% yield) as a yellow solid, after
evaporation of the desired chromatographic fractions. ESMS (m/z): 310 [M+H].
Example 3
5 4-[2-Amino-6-(trifluoromethyl)pyrimidin-4-yl]oxy-3,5-difluoro-benzonitrile
F
F3cyyo~
NYNF~N
NH2
Combine 4-( 4-bromo-2, 6-difl uoro-phenoxy )-6-( trifl uoromethy 1 )pyrimi din-2-
amine (300 mg, 0.8 mmol), zinc cyanide (291 mg, 2.4 mmol),
tetrakis(triphenylphosphine)palladium (0) (188 mg, 162 nmol) in a microwave vial and
10 add N,N-dimethylformamide (6 mL). Seal the vial and heat to 100 °C overnight in a
heating block. Cool the reaction, dilute with water, and extract three times with ethyl
acetate. Combine the organic extracts and dry over sodium sulfate. Filter and evaporate
the resulting filtrate under reduced pressure. Purify the resulting residue by flash
chromatography over silica gel, using a gradient of 5-100% ethyl acetate in hexanes, to
15 afford the title compound (200 mg, 78% yield), after solvent evaporation of the desired
chromatographic fractions. ESMS (m/z): 317 [M+H].
20
Example 4
5-Methyl-4-(trifluoromethyl)-6-(2,4,6-trifluorophenoxy)pyrimidin-2-amine
CH3 F
F3cylyo~
NYN FA)lF
NH2
In a microwave vial, add potassium tert-butoxide (69 mg, 0.6 mmol) to a solution
of 4-chloro-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine (106 mg, 0.5 mmol) and
2,4,6-trifluorophenol (84 mg, 0.6 mmol) in ACN (2.0 mL). Heat the reaction mixture at
120 oc for 30 min in a microwave reactor. Filter the reaction mixture and evaporate the
25 resulting filtrate under a stream of air. Dissolve the resulting residue in 1: 1
5
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dichloromethane/methanol and purify by flash chromatography over silica gel, eluting
with 5-10% ethyl acetate/hexanes, to obtain the title compound (154 mg, 95% yield) as an
off-white solid, after solvent evaporation of the desired chromatographic fractions.
ESMS (m/z): 324 [M+H].
Example 5
4-[2-Amino-5-methyl-6-(trifluoromethyl)pyrimidin-4-yl]oxy-3,5-difluoro-benzonitrile
CH3 F
F3C~O~
N'fNF~N
NH2
Combine 4-((2-amino-5-iodo-6-(trifluoromethyl)pyrimidin-4-yl)oxy)-3,5-
10 difluorobenzonitrile (1.2 g, 2.4 mmol, 90% purity) and trimethylboroxine (2.5 g, 10
mmol, 50% mass) in 1,4-dioxane (25 mL) and then add cesium carbonate (2.4 g, 7.4
mmol) and tetrakis(triphenylphosphine)palladium (0) (580 mg, 0.486855 mmol) and heat
the reaction to 120 oc for 2 h under nitrogen. Cool the reaction mixture and quench with
water (50 mL) and extract with ethyl acetate (50 mL x 2). Combine the organic layers and
15 wash with brine (30 mL x 2). Dry the organic layer over sodium sulfate, filter, and
concentrate under reduced pressure. Purify the crude product by flash silica gel
chromatography initially and then further purify by prep-HPLC (Instrument DD, Method
Column Xtimate C18 150*40 mm*10 urn, Condition water (10 mM NH4HC03)-ACN
Begin B 50%, End B 80%, with a 10 minute gradient time (min) 10,100% B, Hold Time
20 (min) 2, Flow Rate 60 mL/min). The afforded flows are combined, concentrated to
remove most ofCH3CN and then lyophilized to afford 481 mg (60% yield) of the title
compound as a white solid. ES/MS (m/z): 331 (M+H).
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Example 6
4-(2,6-difluorophenoxy)-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine
CH3 F
F3CAO~
NyN FA)
NH2
In a microwave vial, add potassium tert-butoxide (69 mg, 0.6 mmol) to a solution
5 of 4-chloro-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine (106 mg, 0.5 mmol) and 2,6-
difluorophenol (72 mg, 0.6 mmol) in acetonitrile (2.0 mL). Heat the reaction mixture at
120 oc for 30 min in a microwave reactor. Filter the reaction mixture and evaporate
the resulting filtrate under a stream of air. The crude product is purified by reverse phase
chromatography to obtain the title compound (124 mg, 81% yield) as a white
10 solid. ESMS (m/z): 306 [M+H].
IPl Cellular Assay for ECso Determination against hMrgXl by HTRF
Cell plating: HEK293 cells stably expressing the recombinant human MrgXl
receptor are expanded in culture flasks (Corning, Tl50), using growth media containing
15 DMEM with glutamine (GIBCO™, Cat.# 11960-044) supplemented with 10% heatinactivated
FBS (HyCloneTM, Cat. # CH30073), 1% penicillin/streptomycin
(HyClone™, Cat.# SV30010; 10,000 U/mL penicillin; 10,000 IJ.g/mL streptomycin in
0.85% NaCl), 20mM HEPES (GIBCO™, Cat.# 15630122) and 0.3 mg/mL G418
(GIBCO™, Cat.# 11811031). When cell monolayers achieve a level of 80-90%
20 confluence, monolayers are washed once with 10 mL ofDPBS (HyClone™, Cat.#
14190-144), dissociated using TrypLE™ Express enzyme cell dissociation media
(GIBCO™, Cat.# 12605-010), and diluted by addition of 10 mL DPBS. Dissociated cells
are transferred to a sterile 50 mL conical tube, pelleted by centrifugation at 300 x g to
remove the growth and dissociation media, and diluted to 1M cells/mL into DMEM for
25 plating.
IF 1 Potency and Efficacy Determination: Test compounds are dissolved in
DMSO to a concentration of 10 mM and serially diluted in DMSO to obtain a 1 0-point
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concentration response stock dilution plate. Growth media is removed from the cell plate,
and the stock 1 0-point dilution plate is diluted into media and stamped into the cell plate
at a concentration 2x higher than the final test concentration of 30 ~M maximum. The
endogenous agonist BAM8-22 (Tocris-BioScience® Cat. # 1763) is diluted to the EC1s,
5 determined independently at a minimum of n = 3, into the cell plate and incubated at
room temperature for 120 min. Subsequently, halfthe volume each ofanti-IP1 cryptate
and d2-labeled IP1 in lysis buffer, supplied with the IP-One Gq Kit (CisBio Cat. #
62IP APEC) are added to the cell plate to initiate cell lysis, and incubated for 60 min at
room temperature in the dark. At that point, fluorescence is determined at 620 and 665
10 nm (~100 IJ.S following laser excitation).
Data Analysis: Fluorescent ratios are determined as the ratio of the fluorescence
emission at 620 nm over 665 nm and converted to IP1 concentration, using the IP1
standard curve generated in a separate plate, following the manufacturer's instructions.
15 The IP1 concentration is then plotted as a function of compound concentration.
Potentiator potency (ECso) is defined as the compound concentration, in the presence of
the EC1s of the endogenous agonist BAM8-22, resulting in 50% of the increase in IP1
concentration achieved by a saturating concentration ofBAM8-22, and is determined by
using Genedata software (GeneData AG, Basel Switzerland) fitting the following
20 equation to the 10-point CRC, where y is the IP1 concentration determined for a given
compound concentration, [L] denotes the concentration of test compound and Max is the
maximum increase achieved by a saturating concentration ofBAM8-22:
Y=Max*[L]/(ECso + [L])
ECso values are reported as the geometric mean in nM (SEM, n).
25
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Table 1. Relative ECso against hMrgX1 IP-1 for the compounds of Examples 1-6
Example Relative ECso (SEM, n) (nM) Max (Mean ± SEM) ( nM)
1 72 (30, 4) 98 ± 5.3, n=4
2 61 (26, 4) 109 ± 9.2, n=4
3 104 (26, 13) 109 ± 2.5, n=13
4 40 (8, 5) 125 ± 7.9, n=5
5 82,n=1 120
6 38 (3, 3) 103 ± 1.9, n=3
Table 1 shows the relative ECso and the maximum stimulation achieved in the
5 assay for the compounds of Examples 1 to 6, indicating these compounds are potentiators
ofhMrgX1.
In vivo determination of Kp,uu,brain in mice
Unbound brain-to-plasma partition coefficient (Kp,uu,brain) is one of the key
10 pharmacokinetic parameter for evaluating a compound's ability to cross the blood-brain
barrier (BBB). It is typically measured in pre-clinical species using the following
methodology. Kp,uu,brain values indicate the fraction of free drug in plasma that partitions
across the BBB.
Subjects: The subjects for these studies are 12 male C57Bl/6 mice (Envigo, Indianapolis,
15 IN, USA) between 8-10 weeks old at time of test. Mice are housed in groups of 4 in high
density plastic home cages. Food and water is available ad libitum. The rooms are
maintained at 73 °F with 30-70% relative humidity and kept on a light/dark cycle of
0600-1800 h.
Agent: The compound of Example 2 is prepared at 0.3, 1, and 3 mg/ml in the 1% HEC,
20 0.25% TWEEN®80, 0.05% DOWSIL TM vehicle in water. Prepared compound is
sonicated in water bath for 30 min until a suspension is formed. Mice are dosed at 10
ml/kg for a respective 3, 10 or 30 mg/kg dose.
wo 2021/225878 PCT/US2021/030100
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Dosing and Tissue Collection: For this experiment, four mice per dosing group receive
oral dosing of either: 3, 10, or 30 mg/kg of the compound of Example 2. Mice are
euthanized at 2 h post-dosing via C02 asphyxiation, plasma samples are collected via
cardia puncture, and mouse brains removed, weighed, and frozen on dry ice. Blood
5 samples are stored in EDTA tubes on wet ice and centrifuged at 15k rpm for 10 min.
Plasma is collected, plated in a 96-well plate, and frozen at -80 °C.
Pharmacokinetic sampling: Plasma and brain samples obtained are analyzed for
Example 2 using an LC-MS/MS method (Q2 Solutions, Indianapolis, IN, USA). Plasma
samples are extracted using protein precipitation. The lower limit of quantification is 25
10 ng/mL, and the upper limit of quantification is 5000 ng/mL. Brain samples are
homogenized, and the analyte is extracted using protein precipitation. The lower limit of
quantification is 4 ng/g and the upper limit of quantification is 200000 ng/g.
Determination of plasma and brain protein binding: Mouse plasma and brain
homogenate protein binding is determined in vitro using equilibrium dialysis, as
15 described elsewhere [Zamek-Gliszczynski et al., J Pharm Sci, 101:1932-1940, 2012].
The results are reported as fraction unbound in plasma (fu,plasma) and brain (fu,brain) which
are then utilized to calculate Kp,uu,brain as described below. Mouse fu,plasma and fu,brain of
Example 2 are determined to be 0.0421 and 0.0181, respectively.
Analysis and Results: Kp,uu,brain is calculated for each time point from the
20 expression below where individual components are derived from a combination of in
vitro and in vivo measurements carried out as described above:
Cu,brain
Kp,uu,brain = C
u,plasma
Ctotal,brain fu,brain
Ctotal,plasma fu,plasma
where Ctotal,brain, Cu,brain, Ctotal,plasma, and Cu,plasma_are total and unbound brain and plasma
concentrations, and fu,brain and fu,plasma are fractions unbound in brain and plasma,
25 respectively.
wo 2021/225878 PCT/US2021/030100
Time
point
(Hours)
2
2
2
-19-
Table 2. Plasma and brain concentrations of Example 2
post 3, 10, and 30 mg/kg oral dose in mouse.
Unbound Unbound
Total
Total brain brain plasma
Dose plasma
cone. cone. cone.
Group cone.
(mg/kg)
( Ctotal,brain)
( Ctotal,plasma)
(Cu,brain) (Cu,plasma)
(nM)±SD (nM)* (nM)A
(nM)±SD
±SD ±SD
3 972±353 809±140 17.6±6.39 34.1±5.94
10 3900±1800 2770±651 55.4±32.5 117±27.4
9700± 248±
30 13700± 409 ± 101
2410 75.9
4200
Kp,uu,brain
0.506±0.107
0.586±0.116
0.601±.0553
5 *Using mouse fu,brain value of0.0181 and /\mouse fu,plasma value of0.0421, as described
above.
The unbound plasma concentration and unbound brain concentration show dose
related increases in both plasma and brain indicating the compound of Example 2 crosses
the blood brain barrier and has central penetrance at 2 h post-oral administration in mice.
10 There appears to be dose proportionality in plasma & brain exposure across the dose
groups. Mean unbound brain to unbound plasma ratio (Kp,uu-brain) for the compound of
Example 2 ranges from 0.506 ± 0.1 to 0.601 ± 0.0553 (mean± SD, n = 4 per group);
suggesting that an active transport mechanism is not operative in brain tissue in mouse.
WE CLAIM:
1. A compound of the formula:
wherein R1 is hydrogen or methyl; and
R2 is:
, , or
,
or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1 wherein R1 is hydrogen, or a pharmaceutically
acceptable salt thereof.
3. The compound according to claim 1 wherein R1 is methyl, or a pharmaceutically
acceptable salt thereof.
4. The compound according to claim 1 wherein R2 is:
,
or a pharmaceutically acceptable salt thereof.
5. The compound according to claim 1 wherein R2 is:
,
or a pharmaceutically acceptable salt thereof.
6. The compound according to claim 1 wherein R2 is:
,
or a pharmaceutically acceptable salt thereof.
7. The compound according to claim 1 wherein the compound is:
,
or a pharmaceutically acceptable salt thereof.
8. The compound according to claim 7 which is:
.
9. The compound according to claim 1 wherein the compound is:
,
or a pharmaceutically acceptable salt thereof.
10. The compound according to claim 9 which is:
.
11. The compound according to claim 1 wherein the compound is:
,
or a pharmaceutically acceptable salt thereof.
12. The compound according to claim 11 which is:
.
13. The compound according to claim 1 wherein the compound is:
,
or a pharmaceutically acceptable salt thereof.
14. The compound according to claim 13 which is:
.
15. The compound according to claim 1 wherein the compound is:
or a pharmaceutically acceptable salt thereof.
16. The compound according to claim 15 which is:
.
17. The compound according to claim 1 wherein the compound is:
or a pharmaceutically acceptable salt thereof.
18. The compound according to claim 17 wherein the compound is:
.
19. A pharmaceutical composition, comprising a compound or a pharmaceutically
acceptable salt thereof, according to any one of claims 1 to 18 with one or more
pharmaceutically acceptable carriers, diluents, or excipients.
20. A process for preparing a pharmaceutical composition, comprising admixing a
compound or a pharmaceutically acceptable salt thereof according to any one of
claims 1 to 18 with one or more pharmaceutically acceptable carriers, diluents, or
excipients.