PYRAZOLO[1,5-A]PYRIMIDINE-3-CARBOXAMIDE DERIVATIVES USEFUL IN THE TREATMENT OF PSORIASIS AND SYSTEMIC LUPUS ERYTHEMATOSUS

Abstract

The present invention provides a compound of Formula I: wherein R is methyl or ethyl; or a pharmaceutically acceptable salt thereof useful for treating psoriasis or systemic lupus erythematosus.

##STR00001##

Claims

1. A compound of the formula: ##STR00033## wherein R is methyl or ethyl; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 wherein R is methyl; or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 wherein R is ethyl; or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 1 wherein the compound is: ##STR00034## or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 4 wherein the compound is: ##STR00035##

6. The compound according to claim 1 wherein the compound is: ##STR00036## or a pharmaceutically acceptable salt thereof.

7. The compound according to claim 6 wherein the compound is: ##STR00037##

8. A method of treating psoriasis 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.

9. A method of treating systemic lupus erythematosus 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.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. 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.

16. 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

5-(2-Methoxyanilino)-7-(methylamino)-N-[(3R)-1-methyl-2-oxo-pyrrolidin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0076] ##STR00031##

[0077] Scheme 4: A mixture of 5-(2-methoxyanilino)-7-(methylamino)pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (50 g, 159.6 mmol) and (3R)-3-amino-1-methyl-pyrrolidin-2-one; p-toluene sulphonyl salt (60 g, 222 mmol) in pyridine (150 ml) is stirred at ambient temperature for 15 min. Then T3P (1.67 M solution in EtOAc, 185 mL, 309 mmol) is added and the mixture is heated at 80° C. for 3 hours. The reaction mixture is cooled to 60° C. and water (600 mL) is added. The reaction is cooled to ambient temperature and the resulting solid is collected by filtration and washed with water (100 mL).

[0078] The resulting wet solid is dissolved in DMSO (300 mL) and heated to 60° C. Activated charcoal (2 g) is added and stirred at 60° C. for 30 minutes. The mixture is then filtered over diatomaceous earth. Over the filtrated solution, water (300 mL) is added dropwise keeping temperature at 60° C. The mixture is cooled to ambient temperature, and the resulting solid is collected by filtration and washed with water (100 mL). The solid is dried under vacuum to constant weight to give the title compound as a white solid (52 g, 78%). ES/MS m/z 410 (M+H). Chiral SFC: Rt (retention time)=1.63 minutes; SFC Column: Chiralpak AD (4.6×100 mm) 5 μm; isocratic: IPA (0.2% IPAm); Column Temp: 40° C.; Flow Rate: 5.0 mL/min. Optical rotation: [α].sub.D.sup.20=−4.19° (C=0.3, MeOH).

ALTERNATE EXAMPLE 1

5-(2-Methoxyanilino)-7-(methylamino)-N-[(3R)-1-methyl-2-oxo-pyrrolidin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0079] A racemic mixture of 5-(2-methoxyanilino)-7-(methylamino)-N-[rac-1-methyl-2-oxo-pyrrolidin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide is purified via chiral chromatography to give the first eluting enantiomer as the title compound. ES/MS m/z 410 (M+H). Purification conditions: CHIRALPAK® AD-H; Mobile Phase: 10% ACN in MeOH; Flow rate: 30 mL/min; UVW: 225 nm; Retention time: 2.53 minutes. (S enantiomer retention time: 3.78 min).

EXAMPLE 2

5-(2-Ethoxyanilino)-7-(methylamino)-N-[(3R)-1-methyl-2-oxo-pyrrolidin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide

[0080] ##STR00032##

[0081] A racemic mixture of 5-(2-ethoxyanilino)-7-(methylamino)-N-[rac-1-methyl-2-oxo-pyrrolidin-3-yl]pyrazolo[1,5-a]pyrimidine-3-carboxamide is purified via chiral chromatography to give the first eluting enantiomer as the title compound. ES/MS m/z 424 (M+H). Purification conditions: CHIRALPAK® AD-H; Mobile Phase: 40% EtOH in CO.sub.2; Flow rate: 70 g/min; UVW: 260 nm; Retention time: 2.56 minutes. (S enantiomer retention time: 4.28 min).

Binding to TYK2 JH2 by Scintillation Proximity Assay

[0082] The pseudokinase domain (JH2) of human JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) with an N-terminal His.sup.6 tag is expressed in baculovirus and purified by Ni-NTA affinity and size-exclusion chromatography. Yttrium (YSi) His-Tag scintillation proximity assay (SPA) beads (cat #PRNQ0096) are purchased from PerkinElmer Life Sciences. .sup.3H—N-[(1R)-1-[3-[8-Methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]pyridin-2-yl]phenyl]ethyl]-2-(methylsulfonyl) benzamide is synthesized by Quotient Bio with a specific activity of 63 Ci/mmol and a concentration 6.78 μM stored in ethanol (cat #TRQ41678) (See also e.g., J. S. Tokarski, et al., J. Biol. Chem., vol. 290(17), pages 11061-11074 (2015) and R. Moslin, et al., Med. Chem. Commun., vol. 8, pages 700-712 (2017)).

[0083] A 3 fold, 10 point serial dilution of Example 1 is prepared in 100% DMSO (200 nL) and transferred to a 96 well, white, clear bottom, non-binding surface assay plate (Costar 3604) using acoustic liquid handling. Control wells used to determine percent inhibition contained either DMSO (200 nL) or cold, unlabeled inhibitor (200 nL, 10 mM, 200 μM final concentration). His-tagged TYK2 JH2 (20 μL of 7.1 nM) and .sup.3H—N-[(1R)-1-[3-[8-methyl-5-(methylamino)-8H-imidazo[4,5-d]thiazolo[5,4-b]pyridin-2-yl]phenyl]ethyl]-2-(methylsulfonyl) benzamide (50 nM) in assay buffer (50 mM HEPES, pH 7.5, 0.005% Tween-20) are added to the diluted inhibitor. After incubation at room temperature for 2 hours, YSi Copper His-Tag SPA beads (100 μL of 0.5 mg/mL) in phosphate-buffered saline (PBS) containing 0.2% BSA are added to each well. After 15 minutes at room temperature, radioactivity is counted using the Trilux Microbeta. Percent inhibition of radioligand binding at each inhibitor concentration is calculated and fit to the four parameter nonlinear logistic equation using Genedata Screener© to give an IC.sub.50 for the compound of Example 1 of 0.045 μM (±0.016 μM, n=3) and for the compound of Example 2 of 0.040 μM (±0.012 μM, n=4) expressed as GeoMetric means with the standard error of the mean (SEM). This result demonstrates that the compounds of Example 1 and Example 2 bind to the TYK2 JH2 domain in vitro.

Inhibition of IFNα Signaling Through pSTAT1 in TF1 Cells

[0084] TF1 cells (ATCC, CL-2003) are grown in RPMI 1640 (GIBCO) supplemented with 10% dialyzed FBS, 0.1 mg/ml Ampicillin and 2 ng/mL granulocyte macrophage colony stimulating factor. TF1 cells (100 K per well) are seeded in a 96-well poly-D-lysine coated plates in serum-free DMEM and incubated overnight at 37° C. under 5% CO.sub.2. Example 1 is serially diluted in DMSO, added to the cells, and incubated at 37° C. for 1 hr. Cells are then stimulated with 10 ng/ml IFNα2 at 37° C. for 20 minutes. After removing the medium, the cells are lysed in buffer containing Halt protease and phosphatase inhibitor cocktail (Thermo Scientific #78441) at room temperature for 30 minutes. The amount of p-Stat1 (Tyr701) is quantified as light emission at 615 nm using the AlphaLISA SureFire Ultra p-Stat1 (Tyr701) assay kit (Perkin Elmer #ALSU-PST1-A50K) following the vendor's recommended protocol. Percent inhibition at each inhibitor concentration is calculated and fit to the four parameter nonlinear logistic equation using Genedata Screener® to give an IC.sub.50 for the compound of Example 1 of 0.008 μM (±0.001 μM, n=5) and for the compound of Example 2 of 0.010 μM (±0.001 μM, n=4) expressed as GeoMetric means with the standard error of the mean (SEM). This result demonstrates that the compounds of Example 1 and Example 2 are inhibitors of IFNα signaling through pSTAT1 in TF1 cells.

IL23 pSTAT3 AlphaLISA Assay

[0085] IL2-dependent Kit225 cells expressing endogenous IL23 receptors are stably transduced with the Lenti STAT3 Reporter linked to firefly luciferase (SABiosciences CLS-6028L). These cells are used to monitor TYK2 activity by quantifying gene expression caused by STAT3 phosporylation following induction by IL23 in the presence of IL2 using AlphaLISA technology (TGR Biosciences ALSU-TST3-A50K). The cells are grown in RPMI 1640 (Gibco 22400) supplemented with 10% FBS (Invitrogen 10082), 1× Pen/Strep (Gibco 15140-122), 200 ng/ml Puromycin (Sigma P9620), and fresh 10 ng/ml recombinant human IL2 (R&D Systems 202-IL-50).

[0086] For assay preparation, cells are dispensed into Biocoat black poly-d-lysine coated clear bottom 384-well plates (Becton Dickinson Bio-Coat 35-4640) in DMEM (Sigma D5796) at 300,000 cells/well and allowed to incubate overnight at 37° C. Compounds solubilized in DMSO are serially diluted 1:3 to produce a 10-point concentration response curve (final DMSO=0.1%). Cells are pre-incubated with Example 1 for 1 hour at 37° C., then stimulated with IL23 (25 ng/ml final) for 30 minutes. After centrifugation at 2000 rpm for 10 minutes, cell pellets are lysed with a mixture of 1:1 lysis buffer (TGR Biosciences) and Halt Protease & Phosphatase inhibitor cocktail (Thermo Scientific 1861281) for 30 minutes. The AlphaLISA reaction is performed following the vendor's recommended protocol, and the luciferase levels are measured using an Envision plate reader (Perkin Elmer). The relative IC.sub.50 is calculated using a 4-parameter nonlinear logistic equation (GeneData Screener 13.0.5) to give an IC.sub.50 for the compound of Example 1 of 0.009 μM (±0.001 μM, n=4) and for the compound of Example 2 of 0.010 μM (±0.002 μM, n=3) expressed as GeoMetric means with the standard error of the mean (SEM). This result demonstrates that the compounds of Example 1 and Example 2 are inhibitors of IL-23 signaling in a cell-based assay.