(TRIFLUOROMETHYL)PYRIMIDINE-2-AMINE COMPOUNDS

Abstract

The present invention provides a compound of Formula I:

##STR00001## wherein R.sup.1 is hydrogen or methyl; and R.sup.2 is:

##STR00002##

or a pharmaceutically acceptable salt thereof, useful for treating pain, including chronic pain, chronic lower back pain, diabetic peripheral neuropathic pain, and osteoarthritis pain.

Claims

1. A compound of the formula: ##STR00026## wherein R.sup.1 is hydrogen or methyl; and R.sup.2 is: ##STR00027## or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 wherein R.sup.1 is hydrogen, or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 wherein R.sup.1 is methyl, or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1 wherein R.sup.2 is: ##STR00028## or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1 wherein R.sup.2 is: ##STR00029## or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 1 wherein R.sup.2 is: ##STR00030## or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 1 wherein the compound is: ##STR00031## or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 7 which is: ##STR00032##

9. The compound according to claim 1 wherein the compound is: ##STR00033## or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 9 which is: ##STR00034##

11. The compound according to claim 1 wherein the compound is: ##STR00035## or a pharmaceutically acceptable salt thereof.

12. The compound according to claim 11 which is: ##STR00036##

13. The compound according to claim 1 wherein the compound is: ##STR00037## or a pharmaceutically acceptable salt thereof.

14. The compound according to claim 13 which is: ##STR00038##

15. The compound according to claim 1 wherein the compound is: ##STR00039## or a pharmaceutically acceptable salt thereof.

16. The compound according to claim 15 which is: ##STR00040##

17. The compound according to claim 1 wherein the compound is: ##STR00041## or a pharmaceutically acceptable salt thereof.

18. The compound according to claim 17 wherein the compound is: ##STR00042##

19. A method of treating pain in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof.

20. A method of treating chronic pain in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof.

21. A method of treating chronic lower back pain in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof.

22. A method of treating diabetic peripheral neuropathic pain in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof.

23. A method of treating osteoarthritis pain in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1, or a pharmaceutically acceptable salt thereof.

24. A pharmaceutical composition, comprising a compound or a pharmaceutically acceptable salt thereof, according to claim 1 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

25. A process for preparing a pharmaceutical composition, comprising admixing a compound or a pharmaceutically acceptable salt thereof according to claim 1 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

Description

EXAMPLE 1

4-(2,6-Difluorophenoxy)-6-(trifluoromethyl)pyrimidin-2-amine

[0052] ##STR00020##

[0053] In a microwave vial, add potassium tert-butoxide (270 mg, 2.4 mmol) to a solution 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° C. 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 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].

EXAMPLE 2

4-(2,4,6-Trifluorophenoxy)-6-(trifluoromethyl)pyrimidin-2-amine

[0054] ##STR00021##

[0055] In a 500-mL round-bottom flask, add potassium carbonate (26.9 g, 194.7 mmol) to a solution of 2-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 reaction mixture at 80° C. for 16 h. Quench the reaction mixture with water (500 mL) and extract with ethyl acetate (2×500 mL). Wash the combined organic extracts with saturated aqueous NaCl (2×800 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 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

4-[2-Amino-6-(trifluoromethyl)pyrimidin-4-yl]oxy-3,5-difluoro-benzonitrile

[0056] ##STR00022##

[0057] Combine 4-(4-bromo-2,6-difluoro-phenoxy)-6-(trifluoromethyl)pyrimidin-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 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 afford the title compound (200 mg, 78% yield), after solvent evaporation of the desired chromatographic fractions. ESMS (m/z): 317 [M+H].

EXAMPLE 4

5-Methyl-4-(trifluoromethyl)-6-(2,4,6-trifluorophenoxy)pyrimidin-2-amine

[0058] ##STR00023##

[0059] 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° C. for 30 min in a microwave reactor. Filter the reaction mixture and evaporate the resulting filtrate under a stream of air. Dissolve the resulting residue in 1:1 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

[0060] ##STR00024##

[0061] Combine 4-((2-amino-5-iodo-6-(trifluoromethyl)pyrimidin-4-yl)oxy)-3,5-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° C. for 2 h under nitrogen. Cool the reaction mixture and quench with water (50 mL) and extract with ethyl acetate (50 mL×2). Combine the organic layers and wash with brine (30 mL×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 um, Condition water (10 mM NH.sub.4HCO.sub.3)-ACN Begin B 50%, End B 80%, with a 10 minute gradient time (min) 10,100% B, Hold Time (min) 2, Flow Rate 60 mL/min). The afforded flows are combined, concentrated to remove most of CH.sub.3CN and then lyophilized to afford 481 mg (60% yield) of the title compound as a white solid. ES/MS (m/z): 331 (M+H).

EXAMPLE 6

4-(2,6-difluorophenoxy)-5-methyl-6-(trifluoromethyl)pyrimidin-2-amine

[0062] ##STR00025##

[0063] 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 2,6-difluorophenol (72 mg, 0.6 mmol) in acetonitrile (2.0 mL). Heat the reaction mixture at 120° C. 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 solid. ESMS (m/z): 306 [M+H].

[0064] IP1 Cellular Assay for EC.sub.50 Determination Against hMrgX1 by HTRF

[0065] Cell plating: HEK293 cells stably expressing the recombinant human MrgX1 receptor are expanded in culture flasks (Corning, T150), using growth media containing DMEM with glutamine (GIBCO™, Cat. #11960-044) supplemented with 10% heat-inactivated FBS (HyClone™, Cat. #CH30073), 1% penicillin/streptomycin (HyClone™, Cat. #SV30010; 10,000 U/mL penicillin; 10,000 μg/mL streptomycin in 0.85% NaCl), 20 mM HEPES (GIBCO™, Cat. #15530122) and 0.3 mg/mL G418 (GIBCO™, Cat. #11811031). When cell monolayers achieve a level of 80-90% confluence, monolayers are washed once with 10 mL of DPBS (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×g to remove the growth and dissociation media, and diluted to 1M cells/mL into DMEM for plating.

[0066] IP1 Potency and Efficacy Determination: Test compounds are dissolved in DMSO to a concentration of 10 mM and serially diluted in DMSO to obtain a 10-point concentration response stock dilution plate. Growth media is removed from the cell plate, and the stock 10-point dilution plate is diluted into media and stamped into the cell plate at a concentration 2× higher than the final test concentration of 30 μM maximum. The endogenous agonist BAMS-22 (Tocris-BioScience® Cat. #1763) is diluted to the EC.sub.15, determined independently at a minimum of n=3, into the cell plate and incubated at room temperature for 120 min. Subsequently, half the volume each of anti-IP1 cryptate and d2-labeled IP1 in lysis buffer, supplied with the IP-One Gq Kit (CisBio Cat. #62IPAPEC) 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 nm (˜100 μs following laser excitation).

[0067] 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. The IP1 concentration is then plotted as a function of compound concentration. Potentiator potency (EC50) is defined as the compound concentration, in the presence of the EC's of the endogenous agonist BAMS-22, resulting in 50% of the increase in IP1 concentration achieved by a saturating concentration of BAMS-22, and is determined by using Genedata software (GeneData AG, Basel Switzerland) fitting the following 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 of BAMS-22:

Y=Max*[L]/(EC.sub.50+[L])

EC50 values are reported as the geometric mean in nM (SEM, n).

TABLE-US-00001 TABLE 1 Relative EC.sub.50 against hMrgXI IP-1 for the compounds of Examples 1-6 Example Relative EC.sub.50 (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 = l 120 6 38 (3, 3) 103 ± 1.9, n = 3

[0068] Table 1 shows the relative EC50 and the maximum stimulation achieved in the assay for the compounds of Examples 1 to 6, indicating these compounds are potentiators of hMrgX1.

In Vivo Determination of K.SUB.p,uu,brain .in Mice

[0069] Unbound brain-to-plasma partition coefficient (K.sub.p,uu,brain) is one of the key 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. K.sub.p,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, Ind., 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, 0.25% TWEEN®80, 0.05% DOWSIL™ 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.
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 CO2 asphyxiation, plasma samples are collected via cardia puncture, and mouse brains removed, weighed, and frozen on dry ice. Blood samples are stored in EDTA tubes on wet ice and centrifuged at 15 k rpm for 10 min. Plasma is collected, plated in a 96-well plate, and frozen at −80° C.

[0070] Pharmacokinetic sampling: Plasma and brain samples obtained are analyzed for Example 2 using an LC-MS/MS method (Q2 Solutions, Indianapolis, Ind., USA). Plasma samples are extracted using protein precipitation. The lower limit of quantification is 25 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.

[0071] Determination of plasma and brain protein binding: Mouse plasma and brain homogenate protein binding is determined in vitro using equilibrium dialysis, as described elsewhere [Zamek-Gliszczynski et al., J Pharm Sci, 101:1932-1940, 2012]. The results are reported as fraction unbound in plasma (f.sub.u,plasma) and brain (f.sub.u,brain) which are then utilized to calculate K.sub.p,uu,brain as described below. Mouse f.sub.u,plasma and f.sub.u,brain of Example 2 are determined to be 0.0421 and 0.0181, respectively.

[0072] Analysis and Results: K.sub.p,uu,brain is calculated for each time point from the expression below where individual components are derived from a combination of in vitro and in vivo measurements carried out as described above:

[00001]$K_{p,\mathrm{uu},\mathrm{brain}}=\frac{C_{u,\mathrm{brain}}}{C_{u,\mathrm{plasma}}}=\frac{C_{\mathrm{total},\mathrm{brain}}}{C_{\mathrm{total},\mathrm{plasma}}}.Math.\frac{f_{u,\mathrm{brain}}}{f_{u,\mathrm{plasma}}}$

where C.sub.total,brain, C.sub.u,brain, C.sub.total,plasma, and C.sub.u,plasma are total and unbound brain and plasma concentrations, and f.sub.u,brain and f.sub.u,plasma are fractions unbound in brain and plasma, respectively.

TABLE-US-00002 TABLE 2 Plasma and brain concentrations of Example 2 post 3, 10, and 30 mg/kg oral dose in mouse. Unbound Unbound Total brain plasma Total brain plasma conc. conc. Time Dose conc. conc. (C.sub.u,brain) (C.sub.u,plasma) point Group (C.sub.total,brain) (C.sub.total,plasma) (nM)* ± (nM){circumflex over ( )} ± (Hours) (mg/kg) (nM) ± SD (nM) ± SD SD SD K.sub.p,uu,brain 2 3  972 ± 353 809 ± 140 17.6 ± 6.39 34.1 ± 5.94 0.506 ± 0.107 2 10  3900 ± 1800 2770 ± 651  55.4 ± 32.5  117 ± 27.4 0.586 ± 0.116 2 30 13700 ± 4200 9700 ± 2410  248 ± 75.9 409 ± 101 0.601 ± .0553 *Using mouse f.sub.u,brain value of 0.0181 and {circumflex over ( )}mouse f.sub.u,plasma value of 0.0421, as described above.

[0073] 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. 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.