Phenoxyethyl cyclic amine derivatives and their activity as EP4 receptor modulators

Abstract

The present invention provides a compound of the Formula (I): wherein X, R, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, and R.sup.9 are as defined herein, or a pharmaceutically acceptable salt thereof. ##STR00001##

Claims

1. A compound of the formula: ##STR00106## wherein X is: ##STR00107## R is H, methyl, or ethyl; R.sup.1 is methyl, when R.sup.2 is H, and R.sup.1 is H when R.sup.2 is methyl; R.sup.3 is H or F; R.sup.4 is H, F, or methyl; R.sup.5 is OH, methyl, methoxy, or F; and R.sup.6 is H when R.sup.7 is OH, and R.sup.6 is F when R.sup.7 is F; or a pharmaceutically acceptable salt thereof.

2. The compound or salt according to claim 1 wherein R is methyl.

3. The compound or salt according to claim 2 wherein R.sup.4 is F and R.sup.5 is F.

4. The compound or salt according to claim 2 wherein R.sup.4 is methyl and R.sup.5 is methyl.

5. The compound or salt according to claim 1 wherein X is: ##STR00108##

6. The compound or salt according to claim 1 wherein X is: ##STR00109##

7. 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.

Description

EXAMPLE 1

Synthesis of 4-((1S)-1-(((2R*)-1-(2-phenoxyethyl)-2-methylpiperidin-2-carbonyl)amino)ethyl)benzoic acid hydrochloride

(1) ##STR00087##

(2) Dissolve methyl 4-((1S)-1-(((2R*)-1-(2-phenoxyethyl)-2-methylpiperidin-2-carbonyl)amino)ethyl)benzoate (70 mg, 0.16 mmol) in a mixture of THF (1.0 mL) and CH.sub.3OH (1.0 mL). Add a 1.0 N aqueous solution of sodium hydroxide (330 μL, 0.33 mmol), and stir the mixture at room temperature overnight. Concentrate the mixture under reduced pressure to furnish a solid, then add a 4.0 M solution of hydrogen chloride in 1,4-dioxane (2.0 mL, 8.0 mmol) and stir at room temperature for 10 min. Remove the solids by filtration, rinsing through with THF (2 mL). Concentrate the filtrate under reduced pressure to furnish the title compound as an off-white solid (52 mg, 71% yield). Mass spectrum (m/z): 411 (M+H).sup.+.

(3) Prepare the following compounds essentially by the method of Example 1, using the appropriate methyl esters in place of methyl 4-((1S)-1-(((2R*)-1-(2-phenoxyethyl)-2-methylpiperidin-2-carbonyl)amino)ethyl)benzoate:

(4) TABLE-US-00012 MS Ex. Chemical Name Structure (m/z) Note 2 4-((1S)-1-((2R*,6R*)- (1-(2-phenoxyethyl)- 6-methylpiperidin-2- carbonyl)amino)ethyl) benzoic acid hydrochloride 411 (M + H).sup.+ 3 4-(((1S,3R,4R)-2-(2- phenoxyethyl)-2- azabicyclo[2.2.1] heptan-3- carbonyl)aminomethyl) benzoic acid 395 (M + H).sup.+ a, b 4 4-((1S)-1- (((1S,3R,4R)-2-(2- phenoxyethyl)-2- azabicyclo[2.2.1] heptan-3- carbonyl)amino)ethyl) benzoic acid 0 409 (M + H).sup.+ a, b, c 5 4-((1S)-1-(((5R)-6-(2- (4- fluorophenoxy)ethyl)- 6-azaspiro[2.5]octane- 5- carbonyl)amino)ethyl) benzoic acid 441 (M + H).sup.+ d 6 4-((1S)-1-(((5R)-6-(2- phenoxyethyl)-6- azaspiro[2.5]octane-5- carbonyl)amino)ethyl) benzoic acid 423 (M + H).sup.+ d 7 4-((1S)-1- (((1S,4R,6S)-3-(2- phenoxyethyl)-3- azabicyclo[4.1.0] heptane-4- carbonyl)amino)ethyl) benzoic acid 409 (M + H).sup.+ e 8 4-((1S)-1-(((2R)-1-(2- phenoxyethyl)-4,4- difluoropiperidin-2- carbonyl)amino)ethyl) benzoic acid hydrochloride 433 (M + H).sup.+ 9 4-((1S)-1-(((2S)-1-(2- phenoxyethyl)-4,4- difluoropiperidin-2- carbonyl)amino)ethyl) benzoic acid hydrochloride 433 (M + H).sup.+, 455 (M + Na).sup.+ 10 4-((1S)-1-(((2R)-1-(2- (4- fluorophenoxy)ethyl)- 5,5-difluoropiperidin- 2- carbonyl)amino)ethyl) benzoic acid trifluoroacetate 451 (M + H).sup.+ f, g 11 4-((1S)-1-(((2R*)-1- (2-phenoxyethyl)-4,4- dimethylpiperidin-2- carbonyl)amino)ethyl) benzoic acid 425 (M + H).sup.+ c 12 4-((1S)-1-(((2R)-1-(2- phenoxyethyl)-4- oxopiperidin-2- carbonyl)amino)ethyl) benzoic acid trifluoroacetate 411 (M + H).sup.+ d, g 13 4-((1S)-1-(((2R,4S)-1- (2-phenoxyethyl)-4- hydroxypiperidin-2- carbonyl)amino)ethyl) benzoic acid 413 (M + H).sup.+ d 14 4-((1S)-1-(((2R,4S)-1- (2-phenoxyethyl)-4- methoxypiperidin-2- carbonyl)amino)ethyl) benzoic acid 00 427 (M + H).sup.+ d 15 4-((1S)-1-((2R*,5S*)- (1-(2-phenoxyethyl)- 5-hydroxypiperidin-2- carbonyl)amino)ethyl) benzoic acid hydrochloride 01 413 (M + H).sup.+, 435 (M + Na).sup.+ 16 4-((1S)-1-(((2R)-1- (2-(4- fluorophenoxy)ethyl) pyrrolidin-2- carbonyl)amino)ethyl) benzoic acid hydrochloride 02 401 (M + H).sup.+ h 17 4-((1S)-1-(((2R)-1- (2-(4- fluorophenoxy)ethyl) 4,4-difluoropyrrolidin- 2- carbonyl)amino)ethyl) benzoic acid 03 437 (M + H).sup.+ i 18 4-((1S)-1-(((1R)-2-(2- phenoxyethyl)- 1,2,3,4- tetrahydroisoquinolin- 1- carbonyl)amino)ethyl) benzoic acid hydrochloride 04 445 (M + H).sup.+ g a) Instead of acidifying with HCl/1,4-dioxane, neutralize the reaction mixture with 5.0 N aqueous HCl, concentrate under reduced pressure, triturate the material with EtOAc, and decant the solution phase, then remove the solvent to obtain the product. b) Stir the reaction mixture at 70° C. for two hours rather than at room temperature overnight. c) Purify by flash column chromatography on silica gel, eluting with a 0% to 10% CH.sub.3OH/EtOAc gradient. d) Instead of acidifying with HCl/1,4-dioxane, neutralize the reaction mixture to pH 7.0 with 1.0 N aqueous HCl, then extract with EtOAc, wash with saturated aqueous NaCl, dry the organic phase over Na.sub.2SO.sub.4, filter, and concentrate under reduced pressure to obtain the product. e) Instead of acidifying with HCl/1,4-dioxane, neutralize the reaction mixtureto pH 7.0 with 1.0 N aqueous HCl, then concentrate under reduced pressure, triturate the material with 1:1 ethanol/CH.sub.3CN, sonicate, and remove the solids by filtration through a PTFE filter disc. Concentrate the filtrate under reduced pressure to obtain the product. f) Instead of acidifying with HCl/1,4-dioxane, concentrate the reaction mixture under reduced pressure. g) Subject the crude material to reverse-phase chromatography on C18 silica gel, eluting with 0.1% TFA in a CH.sub.3CN/water gradient to obtain the product as a single diastereomer. h) Instead of acidifying with HCl/1,4-dioxane, concentrate under reduced pressure to remove the organic solvents, acidify the reaction mixture to pH 1.0 with 5.0 N aqueous HCl, and isolate the resulting white precipitate by filtration to obtain the product. i) Instead of acidifying with HCl/1,4-dioxane, concentrate under reduced pressure to remove the organic solvents, acidify the reaction mixture to pH 4.0 with 5.0 N aqueous HCl, and isolate the resulting white precipitate by filtration to obtain the product.

EXAMPLE 19

Synthesis of 4-((1S)-1-(((2R)-1-(2-phenoxyethyl)-5,5-difluoropiperidin-2-carbonyl)amino)ethyl)benzoic acid hydrochloride

(5) ##STR00105##

(6) Stir a mixture of methyl 4-((1S)-1-(((2R*)-5,5-difluoropiperidin-2-carbonyl)amino)ethyl)benzoate hydrochloride (200 mg, 0.55 mmol), β-bromophenetole (113 mg, 0.55 mmol), potassium carbonate (229 mg, 1.65 mmol), CH.sub.3CN (1.38 mL), and water (at least 9.9 mg, 0.55 mmol but not more than 29.7 mg, 1.65 mmol) at 70° C. for two days, then at 82° C. for three days. Concentrate the mixture under reduced pressure to furnish a solid. Stir this solid in boiling ethanol, filter, and concentrate the filtrate under reduced pressure. Subject the crude material to reverse-phase chromatography on C18 silica gel, eluting with 0.1% formic acid in a CH.sub.3CN/water gradient, to furnish 161 mg of a mixture of diastereomers of the title compound. Subject 125 mg of this mixture to preparatory supercritical fluid chromatography on a Chiralcel OJ-H 5 μM column, eluting with 0.2% isopropylamine in a 3:1 mixture of supercritical CO.sub.2 to CH.sub.3OH. Isolate the second isomer to elute, and concentrate under reduced pressure. Treat the material with a 4.0 M solution of HCl in 1,4-dioxane (5 mL) and stir for 10 minutes. Concentrate under reduced pressure to furnish the title compound as a white solid (54 mg, 27% yield). Mass spectrum (m/z): 433 (M+H).sup.+, 455 (M+Na).sup.+.

In Vitro Binding to Human EP1, EP2, EP3 and EP4

(7) hEP1 and hEP4 membranes are prepared from recombinant HEK293 cells stably expressing human EP1 (Genbank accession number AY275470) or EP4 (Genbank accession number AY429109) receptors. hEP2 and hEP3 membranes are prepared from HEK293 cells transiently transfected with EP2 (Genbank accession number AY275471) or EP3 (isoform VI: Genbank accession number AY429108) receptor plasmids. Frozen cell pellets are homogenized in homogenization buffer using a Teflon/glass homogenizer. Membrane protein is aliquoted and quick frozen on dry ice prior to storage at −80° C. Homogenization buffer contained 10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.3 mM indomethacin and plus Complete™, with EDTA, obtained from Roche Molecular Biochemicals (Catalog Number 1 697 498).

(8) Kd values for [3H]-PGE.sub.2 binding to each receptor are determined by saturation binding studies or homologous competition. Compounds are tested in a 96-well format using a three-fold dilution series to generate a 10-point curve. Diluted compound is incubated with 20 μg/well EP1, 10 μg/well EP2, 1 ug/well EP3 or 10 to 20 μg/well EP4 membrane for 90 minutes at 25° C. in the presence of 0.3 to 0.5 nM [.sup.3H]-PGE.sub.2 (PerkinElmer, 118 to 180 Ci/mmol). The binding reaction is performed in 200 μL MES buffer (10 mM MES pH 6.0 with KOH, 10 mM MgCl.sub.2 and 1 mM EDTA) using 0.5 mL polystyrene 96-well deep-well plates. Nonspecific binding is calculated by comparing binding in the presence and absence of 2 μM of PGE.sub.2. The membranes are harvested by filtration (TomTek harvester), washed 4 times with cold buffer (10 mM MES pH 6.0 with KOH, 10 mM MgCl.sub.2), dried in a 60° C. oven, and the radioactivity is quantified as counts per minute (CPM) using a TopCount detector. Percent specific binding is calculated as the percent of the binding in the absence of any inhibitor, corrected for binding in the presence of 2 uM of PGE.sub.2. Data are analyzed using a 4-parameter nonlinear logistic equation (ABase Equation 205) as shown: y=(A+((B−A)/(1+((C/x)^D)))) where, y=% specific inhibition, A=bottom of the curve; B=top of the curve; C=relative IC.sub.50=concentration causing 50% inhibition based on the range of the data from top to bottom; D=Hill, Slope=slope of the curve. K.sub.i conversion from IC.sub.50 Values (K.sub.i=IC.sub.50/(1+[L]/K.sub.d) where [L] is the ligand concentration). Results are expressed as the geometric mean±standard deviation; n=number of independent determinations. The standard deviation is calculated by the delta method, being SD.sub.log Ki×geometric mean×ln(10).

(9) The compounds of Examples 1-19 herein are tested essentially as described above and exhibit a K.sub.i value for hEP4 of lower than about 2 μM.

(10) TABLE-US-00013 TABLE 1 In vitro binding of Example 8 to human EP1, EP2, EP3 and EP4 Test hEP1, hEP2, hEP3, hEP4, Compound K.sub.i (nM) K.sub.i (nM) K.sub.i (nM) K.sub.i (nM) Example 8 >12500 956 >14800 3.68 ± 3.32 (n = 1) (n = 1) (n = 1) (n = 6)

(11) The data in table 1 demonstrate the compound of Example 8 binds to hEP4 more strongly than to hEP1, hEP2, and hEP3 indicating selectivity for the hEP4 receptor.

In Vitro Human EP4 Functional Antagonist Activity

(12) Assays are conducted in recombinant HEK293 cells stably expressing human EP4 receptor. The cell lines are maintained by culturing in DMEM with high glucose and pyridoxine hydrochloride (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 mM HEPES, 500 geneticin and 2 mM L-glutamine. Confluent cultures are grown at 37° C. in an atmosphere containing 5% CO.sub.2. Cells are harvested using 2.5% Trypsin-EDTA, suspended in freeze media (FBS with 6% DMSO) at 10.sup.7 cells/mL and aliquots are stored in liquid nitrogen. Just before assay, cells are thawed in DMEM, centrifuged, and resuspended in cAMP buffer.

(13) The inhibition of PGE.sub.2-stimulated cAMP production by EP4 antagonists is measured using HTRF; (Cisbio catalog #62AM4PEB). An aliquot equivalent to 4000 cells is incubated with 50 μL cAMP assay buffer containing EC.sub.80 of PGE.sub.2 (0.188 nM PGE.sub.2 from Sigma, catalog # P5640-10 mg) and antagonists at room temperature for 20 minutes. cAMP assay buffer contains 500 mL HBSS (Hank's Balanced Salt Solution), 0.1% BSA, 20 mM HEPES and 200 μM IBMX (Sigma 15879). CJ-042794 (4-{(1S)-1-[({5-chloro-2-[(4-fluorophenyl)oxy]phenyl}carbonyl)amino]ethyl}benzoic acid) serves as a positive control (See Murase, A., et al., Life Sciences, 82:226-232 (2008)). To measure the cAMP levels, cAMP-d2 conjugate and anti cAMP-cryptate conjugate in lysis buffer are incubated with the treated cells at room temperature for 1 hour. The HTRF signal is detected using an EnVision® plate reader (Perkin-Elmer) to calculate the ratio of fluorescence at 665 nm to 620 nm. The raw data are converted to cAMP amount (pmole/well) using a cAMP standard curve generated for each experiment. Data are analyzed using a 4-parameter nonlinear logistic equation (ABase Equation 205) as shown: y=(A+((B−A)/(1+((C/x)^D)))) where, y=% specific inhibition, A=Bottom of the curve, B=Top of the curve, C=Relative IC.sub.50=concentration causing 50% inhibition based on the range of the data from top to bottom, D=Hill, Slope=slope of the curve. Results are expressed as the geometric mean±standard deviation; n=number of independent determinations. The standard deviation is calculated by the delta method, being SD.sub.log Ki×geometric mean×ln(10).

(14) Following the procedures essentially as described above, the compound of Example 8 has an IC.sub.50 of 1.76±1.51 nM (n=6) measured at human EP4. This demonstrates that the compound of Example 8 is an antagonist of human EP4 in vitro.

In Vitro Antagonist Activity in Human Whole Blood

(15) The inhibitory effects of PGE.sub.2 on LPS-induced TNFα production from macrophages/monocytes are believed to be mediated by EP4 receptors (See Murase, A., et al., Life Sciences, 82:226-232 (2008)). The ability of the compound of Example 8 to reverse the inhibitory effect of PGE.sub.2 on LPS-induced TNFα production in human whole blood is an indicia of functional activity.

(16) Blood is collected from normal volunteer donors into sodium heparin vacutainer tubes. Donors have not taken NSAIDs or celecoxib within 48 hours or glucocorticoids within two weeks of the donation. All tubes/donor are pooled into 50 mL Falcon conical centrifuge tubes and 98 μL/well is distributed into 96-well tissue culture plates (Falcon 3072). Compounds are diluted into DMSO to 100× final and 1 μL/well in triplicate is added to the blood to give 7 point concentration response curves. The blood is pretreated with the compounds at 37° C., in a 5% CO.sub.2 humidified atmosphere, for 30 minutes, after which 1 μL/well of a solution of 1 mg/mL of lipopolysaccharide (LPS) (Sigma 0111:B4) in 0.2 mg/mL bovine serum albumin (BSA)/PBS+/−1 mM PGE.sub.2 (Cayman 14010) is added to give a final LPS concentration of 10 μg/mL+/−10 nM PGE.sub.2. The plates are incubated for 20-24 hours at 37° C. in a 5% CO.sub.2 humidified atmosphere. The plates are centrifuged at 1800×g, 10 minutes at 22° C., in an Eppendorf 5810R centrifuge. Plasma is removed from the cell layer and is transferred to v-bottom polypropylene plates. TNFα levels in 2 μL plasma are quantified by a commercially available enzyme immunoassay (R&D Systems DY210), using Immulon 4 HBX plates (Thermo 3855) and 3,3′,5,5′ tetramethylbiphenyl-4,4′-diamine substrate (KPL 50-76-03). The plates are read at A.sub.450-A.sub.650 on a plate reader (Molecular Devices Versamax) using SOFTmaxPRO (v. 4.3.1) software. IC.sub.50s are calculated using Graphpad Prism (v. 4) nonlinear regression, sigmoidal dose response curve fitting. Results are expressed as the geometric mean±standard deviation; n=number of independent determinations The standard deviation is calculated by the delta method, being SD.sub.log Ki×geometric mean×ln(10).

(17) Following the procedures essentially as described above, the compound of Example 8 has an IC.sub.50 of 0.0782±0.061 uM (n=6) measured at human EP4. This demonstrates that the compound of Example 8 is an EP4 antagonist in the human blood TNFα induction assay.