SUBSTITUTED PYRROLOPYRIDINE-DERIVATIVES

Abstract

The present invention relates to protein-inhibitory substituted pyrrolopyridine derivatives of formula (I) in which A, X, R.sup.1a, R.sup.1b, R.sup.2, R.sup.3a, R.sup.3b, R.sup.4a and R.sup.4b are as defined herein, to pharmaceutical compositions and combinations comprising the compounds according to the invention, and to the prophylactic and therapeutic use of the inventive compounds, respectively to the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of diseases, in particular for neoplastic disorders, respectively cancer or conditions with dysregulated immune responses or other disorders associated with aberrant MAP4K1 signaling, as a sole agent or in combination with other active ingredients. The present invention further relates to the use, respectively to the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of protein inhibitors in benign hyperplasias, atherosclerotic disorders, sepsis, autoimmune disorders, vascular disorders, viral infections, in neurodegenerative disorders, in inflammatory disorders, in atherosclerotic disorders and in male fertility control.

##STR00001##

Claims

1. A compound of formula (I) ##STR00078## wherein R.sup.1a and R.sup.1b together represent a C.sub.3-C.sub.6-cycloalkyl ring; R.sup.2 represents a C.sub.1-C.sub.3-haloalkyl; R.sup.3a and R.sup.3b represent independently hydrogen or a C.sub.1-C.sub.3-alkyl, or R.sup.3a and R.sup.3b together represent a monocyclic 3 to 6-membered cycloalkyl or 4 to 6-membered heterocycloalkyl, wherein said cycloalkyl or heterocycloalkyl is optionally substituted by fluorine, chlorine, methyl, methoxy, hydroxy, or cyano; R.sup.4a and R.sup.4b represent independently hydrogen or a C.sub.1-C.sub.3-alkyl; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof and solvates of the pharmaceutically acceptable salts.

2. The compound of general formula (I) according to claim 1, wherein R.sup.1a and R.sup.1b together represent a C.sub.3-C.sub.5-cycloalkyl ring; R.sup.2 represents a halomethyl or haloethyl group; R.sup.3a and R.sup.3b represent independently hydrogen or a methyl group, or R.sup.3a and R.sup.3b together represent a monocyclic 3 to 5-membered cycloalkyl or 5 or 6-membered heterocycloalkyl, wherein said cycloalkyl or heterocycloalkyl is optionally substituted by fluorine, chlorine, methyl, methoxy, hydroxy, or cyan; and R.sup.4a and R.sup.4b represent independently hydrogen or a methyl group; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

3. The compound of general formula (I) according to claim 1, wherein R.sup.1a and R.sup.1b together represent a cyclopropyl or cyclobutyl ring; R.sup.2 represents a trifluoromethyl; R.sup.3a and R.sup.3b each represents a methyl group; or R.sup.3a and R.sup.3b together represents a cyclopropyl or cyclobutyl ring or a 5 or 6-membered heterocycloalkyl ring having an oxygen atom as heteroatom; and R.sup.4a and R.sup.4b each represents hydrogen; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

4. The compound of general formula (I) according to claim 1, wherein R.sup.1a and R.sup.1b together represent a cyclobutyl ring; R.sup.2 represents a trifluoromethyl; R.sup.3a and R.sup.3b each represents a methyl group, or R.sup.3a and R.sup.3b together represent a cyclopropyl or cyclobutyl ring or a 6-membered heterocycloalkyl ring having an oxygen atom as heteroatom; and R.sup.4a and R.sup.4b each represents hydrogen; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

5. The compound of general formula (I) according to claim 1, wherein R.sup.1a and R.sup.1b together represent a cyclobutyl ring; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof and solvates of the pharmaceutically acceptable salts.

6. The compound of general formula (I) according to claim 1, wherein R.sup.2 represents a trifluoromethyl; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof and solvates of the pharmaceutically acceptable salts.

7. The compound of general formula (I) according to claim 1, wherein R.sup.3a and R.sup.3b each represents a methyl group; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof and solvates of the pharmaceutically acceptable salts.

8. The compound of general formula (I) according to claim 1, wherein R.sup.3a and R.sup.3b together represent a cyclopropyl or cyclobutyl ring or a 6-membered heterocycloalkyl ring having an oxygen atom as hetoeroatom; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

9. The compound of general formula (I) according to claim 1, wherein R.sup.4a and R.sup.4b each represents hydrogen; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof and solvates of the pharmaceutically acceptable salts.

10. The compound according to claim 1, which is selected from the group consisting of: N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-6-oxa-8-azaspiro[3.5]non-7-en-7-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,9-dioxa-4-azaspiro[5.5]undec-3-en-3-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5-oxa-7-azaspiro[2.5]oct-6-en-6-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5,5-dimethyl-5,6-dihydro-4H-1,3-oxazin-2-amine; (+/−)-N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine; (5R)—N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine; (5S)—N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine (single enantiomer 1); N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine (single enantiomer 2); N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,6-dioxa-8-azaspiro[3.5]non-7-en-7-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5,5-dimethyl-5,6-dihydro-4H-1,3-oxazin-2-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5-oxa-7-azaspiro[2.5]oct-6-en-6-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,9-dioxa-4-azaspiro[5.5]undec-3-en-3-amine; N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-6-oxa-8-azaspiro[3.5]non-7-en-7-amine; (+/−)-N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine; and N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,6-dioxa-8-azaspiro[3.5]non-7-en-7-amine.

11. (canceled)

12. (canceled)

13. A pharmaceutical composition comprising the compound of general formula (I) according to claim 1 and one or more pharmaceutically acceptable excipients.

14. A pharmaceutical combination comprising: one or more of the compounds of general formula (I) according to claim 1, and one or more selected from the group consisting of: one or more pharmaceutically active anti-cancer compounds, and one or more pharmaceutically active immune checkpoint inhibitors.

15. A pharmaceutical combination according to claim 14, wherein the pharmaceutically active immune checkpoint inhibitor is an antibody.

16. A method of treating a disease, condition, or disorder comprising administering the compound of general formula (I) according to claim 1.

17. A method of preparing a pharmaceutical composition, comprising mixing the compound of general formula (I) according to claim 1 with one or more pharmaceutically acceptable excipients.

18. The method of claim 16, wherein the disease, condition, or disorder is selected from the group consisting of: cancer, a condition associated with dysregulated immune responses, a disorder associated with aberrant MAP4K1 signaling, or tumors associated with aberrant MAP4K1 signaling.

19. The method of claim 16, wherein the disease, condition, or disorder is selected from the group consisting of: benign hyperplasias, atherosclerotic disorders, sepsis, autoimmune disorders, vascular disorders, viral infections, neurodegenerative disorders, and inflammatory disorders.

20. A method of controlling male fertility comprising administering a compound of general formula (I) according to claim 1.

21. The compound of general formula (I) according to claim 2, wherein R.sup.1a and R.sup.1b together represent a cyclobutyl ring; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

22. The compound of general formula (I) according to claim 3, in which R.sup.1a and R.sup.1b together represent a cyclobutyl ring; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

23. The compound of general formula (I) according to claim 2, in which R.sup.2 represents a trifluoromethyl; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

24. The compound of general formula (I) according to claim 2, in which R.sup.3a and R.sup.3b each represents a methyl group; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

25. The compound of general formula (I) according to claim 2, in which R.sup.3a and R.sup.3b together represent a cyclopropyl or cyclobutyl ring or a 6-membered heterocycloalkyl ring having an oxygen atom as heteroatom; and polymorphs, enantiomers, diastereomers, racemates, tautomers, solvates, pharmaceutically acceptable salts thereof, and solvates of the pharmaceutically acceptable salts.

Description

EXAMPLE 1

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-6-oxa-8-azaspiro[3.5]non-7-en-7-amine

[0480] ##STR00064##

[0481] N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-N′-{[1-(hydroxymethyl)cyclobutyl]methyl}thiourea (Intermediate 16, 90.0 mg, 166 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (63.8 mg, 333 μmol), and trimethylamine (70 μL, 500 μmol) were dissolved in acetonitrile (1 mL) and stirred overnight at 40° C. Water was added, and the mixture extracted with ethyl acetate. The organic layers were dried over sodium sulfate, filtered and the solvent removed under vacuum. The crude product was purified by silica gel chromatography and digested in hexanes/diethyl ether to yield the title compound (52 mg, 59% yield).

[0482] LC-MS (Method 4): R.sub.t=1.36 min; MS (ESIpos): m/z=507 [M+H].sup.+

[0483] .sup.1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 11.93 (d, 1H), 8.98 (br s, 1H), 8.04 (d, 1H), 7.50 (d, 1H), 7.48 (br s, 2H), 6.23 (d, 1H), 4.13 (s, 2H), 3.30 (m, 2H), 2.66 (m, 4H), 2.08-1.78 (m, 8H).

EXAMPLE 2

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,9-dioxa-4-azaspiro[5.5]undec-3-en-3-amine

[0484] ##STR00065##

[0485] N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-N′-{[4-(hydroxymethyl)oxan-4-yl]methyl}thiourea (Intermediate 18, 90.0 mg, 158 μmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (60.5 mg, 315 μmol), and triethylamine (66 μl, 470 μmol) were dissolved in acetonitrile (950 μL) and stirred overnight at 40° C. Water was added, and the mixture extracted with ethyl acetate. The organic layers were dried over sodium sulfate, filtered and the solvent removed under vacuum. The crude product was purified by silica gel chromatography and digested in hexanes/diethyl ether to yield the title compound (39 mg, 44% yield).

[0486] LC-MS (Method 4): R.sub.t=1.22 min; MS (ESIpos): m/z=537 [M+H].sup.+

[0487] .sup.1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 11.93 (d, 1H), 9.02 (br s, 1H), 8.04 (d, 1H), 7.53 (br s, 2H), 7.50 (d, 1H), 6.25 (d, 1H), 4.06 (s, 2H), 3.60 (m, 4H), 3.26 (br s, 2H), 2.66 (br t, 4H), 2.05-1.90 (m, 2H), 1.43 (br s, 4H).

EXAMPLE 3

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5-oxa-7-azaspiro[2.5]oct-6-en-6-amine

[0488] ##STR00066##

[0489] N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclobutyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-5-oxa-7-azaspiro[2.5]oct-6-en-6-amine (Intermediate 20, 119 mg, 191 μmol) and trifluoroacetic acid (370 μl, 4.8 mmol) were dissolved in dichloromethane (2 mL) and stirred at room temperature for 5 h. Acetonitrile (2 mL) and aqueous ammonia (33%, 1 mL) were added, and the mixture stirred for 1 h. The solvent was removed under vacuum and the residue purified by preparative HPLC to yield the title compound (16 mg, 16% yield).

[0490] LC-MS (Method 4): R.sub.t=1.29 min; MS (ESIpos): m/z=493 [M+H].sup.+

[0491] .sup.1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 11.93 (d, 1H), 8.98 (br s, 1H), 8.05 (d, 1H), 7.55 (br s, 2H), 7.49 (d, 1H), 6.24 (d, 1H), 4.02 (s, 2H), 3.20 (br s, 2H), 2.66 (br t, 4H), 2.06-1.88 (m, 2H), 0.61 (br s, 2H), 0.56 (br s, 2H).

EXAMPLE 4

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5,5-dimethyl-5,6-dihydro-4H-1,3-oxazin-2-amine

[0492] ##STR00067##

[0493] N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclobutyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-5,5-dimethyl-5,6-dihydro-4H-1,3-oxazin-2-amine (Intermediate 22, 124 mg, 198 μmol) and trifluoroacetic acid (380 μl, 5.0 mmol) were dissolved in dichloromethane (2 mL) and stirred at room temperature for 5 h. Acetonitrile (2 mL) and aqueous ammonia (33%, 1 mL) were added, and the mixture stirred for 1 h. The solvent was removed under vacuum and the residue purified by preparative HPLC to yield the title compound (20 mg, 20% yield).

[0494] LC-MS (Method 4): R.sub.t=1.33 min; MS (ESIpos): m/z=495 [M+H].sup.+

[0495] .sup.1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 11.93 (d, 1H), 9.00 (br s, 1H), 8.05 (d, 1H), 7.55 (br s, 2H), 7.50 (d, 1H), 6.25 (d, 1H), 3.87 (br s, 2H), 3.07 (br s, 2H), 2.66 (br t, 4H), 2.05-1.90 (m, 2H), 0.96 (s, 6H).

EXAMPLE 5

(+/−)-N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine

[0496] ##STR00068##

[0497] To a solution of (+/−)-N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclopropyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine (600 mg, 0.94 mmol, intermediate 35) in dichloromethane (4.0 mL) was added trifluoroacetic acid (4.0 mL). The reaction mixture was stirred at room temperature overnight, at which time the mixture was basificed to pH >10 with 2M sodium hydroxide and ethyl acetate was added. The layers were separated and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and evaporated. The crude material was dissolved in acetonitrile (10 mL) and treated with a 25% aqueous solution of ammonia (5 mL). The resulting solution was stirred for 1 hour and then purified by preparative HPLC to afford the title compound (284 mg, 59% yield).

[0498] LC-MS (Method 4): R.sub.t=1.15 min; MS (ESIpos): m/z=509 [M+H].sup.+

[0499] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 5 ppm 1.18 (br s, 2H), 1.30-1.37 (m, 2H), 1.65-1.82 (m, 2H), 3.41-3.50 (m, 1H), 3.60 (d, 1H), 3.74-3.85 (m, 2H), 4.10 (q, 2H), 6.26 (d, 1H), 7.53 (d, 1H), 7.57 (br s, 1H), 8.04 (d, 1H), 9.07 (br s, 1H), 11.91 (br d, 1H)

[0500] The title compound was separated into its enantiomers by preparative chiral SFC to give stereoisomer 1 (76 mg, see example 6), stereoisomer 2 (58 mg, see example 7). For the isolation of stereoisomer 1, and stereoisomer 2 the following method was used.

[0501] Analytical chiral SFC method:

[0502] Instrument: Agilent: 1260, Aurora SFC-Module; Column: Chiralpak IC 5μ 100×4.6 mm; Eluent A: CO.sub.2; Eluent B: Ethanol+0.1 Vol-% NH.sub.4OH (32%); Isocratic: 20% B; Flowrate: 4 ml/min; Temperature: 37.5° C.; Pressure: 100 bar; UV: 254 nm Preparative chiral SFC method:

[0503] Instrument: Sepiatec: Prep SFC100; Column: Chiralpak IC 5μ 250×30 mm; Eluent A: CO.sub.2; Eluent B: Ethanol+0.2 Vol-% NH.sub.4OH (32%); Isocractic: 20% B; Flowrate: 100 ml/min; Temperature: 40° C.; Pressure: 150 bar; UV: 254 nm

EXAMPLE 6, Example 7

(5R)—N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine

(5S)—N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine

EXAMPLE 6

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine (single enantiomer 1)

[0504] ##STR00069##

[0505] For the preparation of the title compound and separation into its isomers, see example 5. Analytical chiral HPLC (method, see example 5): Rt=2.10 min, ee=97.7%

EXAMPLE 7

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine (single enantiomer 2)

[0506] ##STR00070##

[0507] For the preparation of the title compound and separation into its isomers, see example 5. Analytical chiral HPLC (method, see example 5): Rt=2.62 min, ee=96.2%

EXAMPLE 8

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,6-dioxa-8-azaspiro[3.5]non-7-en-7-amine

[0508] ##STR00071##

[0509] In analogy to Example 5, N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclopropyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-2,6-dioxa-8-azaspiro[3.5]non-7-en-7-amine (175 mg, 280 μmol, intermediate 38) was treated with trifluoroacetic acid (1.0 mL) in dichloromethane (2.0 mL) to afford after preparative HPLC purification the title compound (46 mg, 32% yield).

[0510] LC-MS (Method 4): R.sub.t=1.10 min; MS (ESIpos): m/z=495 [M+H].sup.+

[0511] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 5 ppm 1.18 (br s, 2H), 1.31-1.36 (m, 2H), 3.60 (s, 2H), 4.39-4.45 (m, 6H), 6.24 (d, 1H), 7.41 (br s, 2H), 7.53 (s, 1H), 8.03 (d, 1H), 11.91 (br s, 1H)

EXAMPLE 9

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5,5-dimethyl-5,6-dihydro-4H-1,3-oxazin-2-amine

[0512] ##STR00072##

[0513] In analogy to Example 5, N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclopropyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-5,5-dimethyl-5,6-dihydro-4H-1,3-oxazin-2-amine (280 mg, 458 μmol, intermediate 40) was treated with trifluoroacetic acid (2.0 mL) in dichloromethane (2.0 mL) to afford after preparative HPLC purification the title compound (82 mg, 37% yield).

[0514] LC-MS (Method 4): R.sub.t=1.28 min; MS (ESIpos): m/z=482 [M+H].sup.+

[0515] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 5 ppm 0.96 (br s, 6H), 1.18 (br s, 2H), 1.30-1.36 (m, 2H), 3.10 (br s, 2H), 3.87 (br s, 2H), 6.27 (d, 1H), 7.53 (d, 1H), 7.58 (br s, 1H), 8.04 (d, 1H), 9.01 (br s, 1H), 11.91 (brd, 1H)

EXAMPLE 10

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-5-oxa-7-azaspiro[2.5]oct-6-en-6-amine

[0516] ##STR00073##

[0517] In analogy to Example 5, N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclopropyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-5-oxa-7-azaspiro[2.5]oct-6-en-6-amine (175 mg, 288 μmol, intermediate 42) was treated with trifluoroacetic acid (1.0 mL) in dichloromethane (2.0 mL) to afford after preparative HPLC purification the title compound (28 mg, 19% yield).

[0518] LC-MS (Method 4): R.sub.t=1.25 min; MS (ESIpos): m/z=479.7 [M+H].sup.+

[0519] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 0.59 (br d, 4H), 1.18 (br s, 2H), 1.31-1.37 (m, 2H), 3.21 (br s, 2H), 4.02 (s, 2H), 6.25 (d, 1H), 7.53 (s, 1H), 7.57 (br s, 1H), 8.04 (d, 1H), 8.99 (br s, 1H), 11.91 (br s, 1H)

EXAMPLE 11

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,9-dioxa-4-azaspiro[5.5]undec-3-en-3-amine

[0520] ##STR00074##

[0521] To a solution of N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclopropyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-2,9-dioxa-4-azaspiro[5.5]undec-3-en-3-amine (190 mg, 291 μmol, intermediate 44) in dichloromethane (2.0 mL) was added trifluoroacetic acid (1.0 mL). The reaction mixture was stirred at room temperature overnight, at which time the mixture was basificed to pH >10 with 2M sodium hydroxide and ethyl acetate was added. The layers were separated and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and evaporated. The crude material was dissolved in acetonitrile (10 mL) and treated with a 25% aqueous solution of ammonia (5 mL). The resulting solution was stirred for 1 hour and then purified by preparative HPLC to afford the title compound (38 mg, 24% yield).

[0522] LC-MS (Method 4): R.sub.t=1.16 min; MS (ESIpos): m/z=524 [M+H].sup.+

[0523] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.18 (br s, 2H), 1.31-1.35 (m, 2H), 1.44 (br s, 4H), 3.28 (br d, 2H), 3.54-3.67 (m, 4H), 4.07 (br s, 2H), 6.26 (d, 1H), 7.53 (s, 1H), 7.57 (br s, 1H), 8.04 (d, 1H), 9.03 (br s, 1H), 11.91 (s, 1H)

EXAMPLE 12

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclopropyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-6-oxa-8-azaspiro[3.5]non-7-en-7-amine

[0524] ##STR00075##

[0525] In analogy to Example 5, N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclopropyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-6-oxa-8-azaspiro[3.5]non-7-en-7-amine (280 mg, 450 μmol, intermediate 46) was treated with trifluoroacetic acid (2.0 mL) in dichloromethane (2.0 mL) to afford after preparative HPLC purification the title compound (68 mg, 30% yield).

[0526] LC-MS (Method 4): R.sub.t=1.31 min; MS (ESIpos): m/z=494 [M+H].sup.+

[0527] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.18 (br s, 2H), 1.31-1.36 (m, 2H), 1.78-2.02 (m, 6H), 3.31 (br s, 2H), 4.14 (s, 2H), 6.26 (d, 1H), 7.53 (d, 1H), 8.04 (d, 1H), 9.01 (br s, 1H), 11.91 (d, 1H)

EXAMPLE 13

(+/−)-N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine

[0528] ##STR00076##

[0529] In analogy to Example 5, (+/−)-N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclobutyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-2,7-dioxa-9-azaspiro[4.5]dec-8-en-8-amine (95.0 mg, 146 μmol, intermediate 48) was treated with trifluoroacetic acid (0.5 mL) in dichloromethane (1.0 mL) to afford after preparative HPLC purification the title compound (33 mg, 41% yield).

[0530] LC-MS (Method 4): R.sub.t=1.20 min; MS (ESIpos): m/z=523 [M+H].sup.+

[0531] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.65-1.81 (m, 2H), 1.89-2.06 (m, 2H), 2.62-2.70 (m, 4H), 3.44 (br d, 1H), 3.60 (d, 1H), 3.74-3.85 (m, 2H), 4.10 (s, 2H), 6.24 (d, 1H), 7.50 (d, 1H), 7.53 (br s, 1H), 8.05 (d, 1H), 9.07 (br s, 1H), 11.93 (br d, 1H)

EXAMPLE 14

N-[3,5-difluoro-4-({3-[1-(trifluoromethyl)cyclobutyl]-1H-pyrrolo[2,3-b]pyridin-4-yl}oxy)phenyl]-2,6-dioxa-8-azaspiro[3.5]non-7-en-7-amine

[0532] ##STR00077##

[0533] In analogy to Example 5, N-{3,5-difluoro-4-[(3-[1-(trifluoromethyl)cyclobutyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl}-2,6-dioxa-8-azaspiro[3.5]non-7-en-7-amine (175 mg, 274 μmol, intermediate 51) was treated with trifluoroacetic acid (1.0 mL) in dichloromethane (2.0 mL) to afford after preparative HPLC purification the title compound (38 mg, 26% yield).

[0534] LC-MS (Method 4): R.sub.t=1.16 min; MS (ESIpos): m/z=509 [M+H].sup.+

[0535] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.87-2.05 (m, 2H), 2.61-2.70 (m, 4H), 3.59 (s, 2H), 4.38-4.46 (m, 6H), 6.22 (d, 1H), 7.38 (br s, 1H), 7.50 (s, 1H), 8.04 (d, 1H), 11.94 (br s, 1H)

Experimental Section—Biological Assays

[0536] Biological in Vitro Assays

[0537] The in vitro activity of the compounds of the present invention can be demonstrated in the following assays:

[0538] The example testing experiments described herein serve to illustrate the present invention and the invention is not limited to the examples given.

[0539] Biological Evaluation

[0540] In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.

[0541] Demonstration of the activity of the compounds of the present invention may be accomplished through in vitro and in vivo assays that are well known in the art. For example, to demonstrate the efficacy of a pharmaceutical agent to inhibit and be selective against e.g. TBK1 the following assays may be used.

[0542] MAP4K1 Binding Competition Assay

[0543] The ability of the compounds of the present invention to inhibit the binding of an Alexa647-labelled ATP-competitive kinase inhibitor to a Glutathione-S-transferase-(GST-) fusion protein was quantified employing the TR-FRET-based binding competition assay as described in the following paragraphs.

[0544] A recombinant fusion protein of N-terminal GST and full-length human, expressed by baculovirus infected SF9 insect cells and purified by Glutathione Sepharose affinity chromatography, was used as GST-fusion protein. Tracer 222 from Invitrogen (catalogue no. PR9198A) was used as Alexa647-labelled ATP-competitive kinase inhibitor.

[0545] For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 3 μL solution of Tracer 222 (25 nM=>final concentration in 5 μL assay volume is 15 nM) in aqueous assay buffer [25 mM Tris/HCl pH 7.5, 10 mM MgCl.sub.2, 5 mM β-glycerolphosphate, 2.5 mM dithiothreitol, 0.5 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid [EGTA], 0.5 mM sodium ortho-vanadate, 0.01% (w/v) bovine serum albumin [BSA], 0.005% (w/v) Pluronic F-127 (Sigma)] were added. Then the binding competition was started by the addition of 2 μL of a solution of the GST-fusion protein (2.5 nM=>final cone, in the 5 μL assay volume is 1 nM) and of Anti-GST-Tb (1.25 nM=>final cone, in the 5 μL assay volume is 0.5 nM), a Lumi4®-Tb Cryptate-conjugated anti-GST-antibody from Cisbio Bioassays (France), in assay buffer.

[0546] The resulting mixture was incubated 30 min at 22° C. to allow the formation of a complex between the Tracer 222, the fusion protein and Anti-GST-Tb. Subsequently the amount of this complex was evaluated by measurement of the resonance energy transfer from the Tb-cryptate to the Tracer 222. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Pherastar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of the complex. The data were normalised (assay reaction without inhibitor=0% inhibition, all other assay components but GST-fusion protein=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100 fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated using Genedata Screener™ software.

[0547] ROCK-II Kinase Assay

[0548] ROCK-II-inhibitory activity of compounds of the present invention was quantified employing the ROCK-II assay as described in the following paragraphs. In essence, the enzyme activity is measured by quantification of the adenosine-di-phosphate (ADP), which is generated as a co-product of the enzyme reaction, via the “ADP-Glo™ Kinase Assay” kit from the company Promega. This detection system works as follows: In a first step the adenosine-tri-phosphate (ATP) not consumed in the kinase reaction is quantitatively converted to cAMP employing an adenylate cyclase (“ADP-Glo-reagent”), then the adenylate cyclase is stopped and the ADP generated in the kinase reaction converted to ATP which generates in a luciferase-based reaction a glow-luminescence signal (“Kinase Detection Reagent”).

[0549] Recombinant N-terminal His6-tagged human ROCK-II (amino acids 11-552), expressed by baculovirus infected SF21 insect cells and purified via Ni.sup.2+-NTA-agarose affinity chromatography, was purchased from Eurofins (product no. 14-451-K) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK (C-terminus in amide form) was used which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany).

[0550] For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a white 1536 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μl of a solution of ROCK-II in aqueous assay buffer [50 mM TRIS/HCl pH 7.5, 10 mM MgCl.sub.2, 0.1 mM EGTA, 0.001% (w/v) bovine serum albumin] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μl of a solution of ATP (16.7 μM=>final cone, in the 5 μl assay volume is 10 μM) and peptide substrate (16.7 μM=>final cone, in the pi assay volume is 10 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of ROCK-II was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, a typical concentration is about 5 nM. The reaction was stopped by the addition of 2.5 μl of “ADP-Glo-reagent” (1:1, 5 fold diluted) and the resulting mixture was incubated at 22° C. for 1 h to convert the ATP not consumed in the kinase reaction completely to cAMP. Subsequently 2.5 μl of the “kinase detection reagent” (1.2 fold more concentrated than recommended by the producer) were added, the resulting mixture was incubated at 22° C. for 1 h and then the luminescence measured with a suitable measurement instrument (e.g. Viewlux™ from Perkin-Elmer). The amount of emitted light was taken as a measure for the amount of ADP generated and thereby for the activity of the ROCK-II.

[0551] The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.1 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100 fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC.sub.50 values were calculated using Genedata Screener™ software.

TABLE-US-00002 TABLE 1 Measured IC.sub.50 values of compounds regarding MAP4K1 inhibition, ROCK- II inhibition and the selectivity ratio between the two inhibition values. MAP4K1 ROCK-II Ratio Example IC.sub.50 [nM] IC.sub.50 [μM] MAP4K1/ROCK-11 1 8.5 5.6 659 2 4.2 1.9 452 3 4.0 1.6 404 4 6.2 4.1 658 5 5.9 0.40  68 6 7.9 0.52  66 7 5.5 0.51  93 8 5.0 0.50 100 9 8.4 0.94 112 10 4.8 0.65 135 11 3.9 0.56 143 12 7.4 1.4 191 13 6.2 1.2 193 14 6.9 1.3 181

[0552] TBK1 High ATP Kinase Assay

[0553] TBK1-inhibitory activity of compounds of the present invention at a high ATP concentration after preincubation of enzyme and test compounds was quantified employing the TR-FRET-based TBK1 assay as described in the following paragraphs.

[0554] Recombinant full-length N-terminally His-tagged human TBK1, expressed in insect cells and purified by Ni-NTA affinity chromatography, was purchased from Life Technologies (Cat. No PR5618B) and used as enzyme. As substrate for the kinase reaction biotinylated peptide biotin-Ahx-GDEDFSSFAEPG (C-terminus in amide form) was used which can be purchased e.g. form the company Biosyntan (Berlin-Buch, Germany).

[0555] For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into either a black low volume 384 well microtiter plate or a black 1536 well microtiter plate (both Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of TBK1 in aqueous assay buffer [50 mM HEPES pH 7.0, 10 mM MgCl.sub.2, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumine, 0.01% (v/v) Nonidet-P40 (Sigma), protease inhibitor mixture (“Complete w/o EDTA” from Roche, 1 tablet per 5 mL)] were added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 1.67 mM=>final cone, in the 5 μL assay volume is 1 mM) and substrate (1.67 μM=>final cone, in the 5 μL assay volume is 1 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of TBK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.002-0.004 μg/mL. The reaction was stopped by the addition of 3 μL of a solution of TR-FRET detection reagents (0.33 μM streptavidine-XL665 [Cisbio Bioassays, Codolet, France], 2.5 nM anti-phosho-Serine antibody [Merck Millipore, “STK antibody”, cat. #35-002] and 1.25 nM LANCE EU-W1024 labeled anti-mouse IgG antibody [Perkin-Elmer, product no. AD0077]) in an aqueous EDTA-solution (167 mM EDTA, 0.13% (w/v) bovine serum albumin in 100 mM HEPES/NaOH pH 7.5).

[0556] The resulting mixture was incubated 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents.

[0557] Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm was measured in a TR-FRET reader, e.g. a Pherastar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition). Usually the test compounds were tested on the same microtiterplate in 11 different concentrations in the range of 20 μM to 0.07 nM (20 μM, 5.7 μM, 1.6 μM, 0.47 μM, 0.13 μM, 38 nM, 11 nM, 3.1 nM, 0.9 nM, 0.25 nM and 0.07 nM, the dilution series prepared separately before the assay on the level of the 100 fold concentrated solutions in DMSO by serial dilutions, exact concentrations may vary depending pipettors used) in duplicate values for each concentration and IC50 values were calculated using Genedata Screener™ software.

TABLE-US-00003 TABLE 2 Measured IC.sub.50 values of compounds regarding TBK1 inhibition as selectivity assay TBK1 Example IC.sub.50 [μM] 1 >20 2 >20 3 >20 4 >20 5 >20 6 >20 7 >20 8 >20 9 >20 10  >20 11  >20 12  >20 13  >20 14  >20

[0558] Phosphorylation Assay in Human Cell Line

[0559] Phosphorylation assays were carried out in Jurkat E6.1 cells from American Type Culture Collection (ATCC) stably overexpressing human FLAG-tagged SLP-76 (proprietary). Cultured cells were kept in RPMI 1640 medium supplemented with 1% FCS at a cell density of 2×10e6/mL 24 h prior compound testing. Starved cells were transferred to a 384 well format plate at a cell density of 140.000 cells/well and simultaneously treated with 1 μg/mL a-CD3 antibody (clone OKT3. ebioscience #16-0037-85) and 4 μg/mL anti-IgG crosslinking antibody (Invitrogen goat anti-mouse IgG (H+L) 2 #31160) together with the test compound for 30 min at 37° C. Applied compounds were tested at either fixed concentration of 10 μmol/L and 20 μmol/L or in a 8 point dose response titration of increase compound concentration with 10 nmol/L. 50 nmol/L. 100 nmol/L. 500 nmol/L. 1 μmol/L. 5 μmol/L. 10 μmol/L and 20 μmol/L in triplicates. The cells were washed once in phosphate-buffered saline (pH 7.4). The detection of pSer376-SLP76 levels in the proprietary Jurkat cell lines was carried out utilizing an adapted protocol of the HTRF pSLP76 Assay (Cisbio #63ADK076PEG). Cells were lysed using 4 μl of the supplemented lysis buffer (Cisbio #63ADK076PEG) for 60 min at room temperature. Subsequently 4 μl of the premixed antibody solution (Cisbio #63ADK076PEG) was added and incubated over night at room temperature. Read-out and analyses was carried out using a Pherastar and the MARS software (BMG Labtechnologies, Offenburg, Germany).

[0560] As control for maximal effect (max control which represent the maximally possible inhibition of pSer376-SLP-76 by a test compound) cells with no a-CD3 (clone OKT3. ebioscience #16-0037-85) and no test compound treatment were used. Cells with a-CD3 treatment only were used as negative control (min control, which represent the minimally possible inhibition of pSer376-SLP-76 by a test compound).

TABLE-US-00004 TABLE 3 Measured IC.sub.50 values / % amount of pSer376-SLP-76 of compound Example IC.sub.50 [μM] 1 2 1.3 3 1.2 4 3.0 5 0.94 6 1.3 7 0.89 8 0.33 9 2.7 10 2.0 11 1.1 12 8.5 13 1.5 14 0.51

[0561] Stimulation of IFNg Production from Human Primary Peripheral Blood Mononuclear Cells (PBMCs)

[0562] The effect of the compound in the activation of human T cells was tested by measuring the production of the proinflammatory cytokine IFNg in vitro. Fresh human PBMCs were isolated and activated in vitro with coated a-CD3 (clone OKT3. ebioscience #16-0037-85. plate-bound). Concentration of a-CD3 was titrated in order to obtain a sub-optimal activation of PBMCs (1×10.sup.6 PBMCs/mL). Cells were activated with a-CD3 and 1 μmol/L PGE2 for 22 hours in the presence of the compounds and the supernatant of the culture was isolated and tested for IFNg concentration. Applied compounds were tested at either fixed concentration of 200 nmol/L or in a 6 point dose response titration of increase compound concentration from 12 nmol/L to 3 μmol/L in triplicates. IFNg concentration was determined by ELISA (Opt EIA human IFNg ELISA BD #555142). Plate was coated with a-IFNg overnight. The plates were washed 3 times and the supernatant from the PBMCs culture was added to the wells and incubated for 2 hours. Plates were washed and detection antibody and the SAv-HRP was added for 1 h. Plates were washed and the substrate was added until the standard turns blue. The reaction is stopped by adding 50 μL 2N H2SO4. Absorbance was measured with a TECAN Reader at 450 to 570 nm. Concentration of IFNg was calculated from the absorbance using standards of known concentration.

[0563] FIG. 1 shows the efficacy of a selected example in IFNγ production (Human primary peripheral blood mononuclear cells)