IMIDAZOPYRAZINE SYK INHIBITORS

Abstract

Certain imidazopyrazines and pharmaceutical compositions thereof are provided herein. Methods of treating patients suffering from certain diseases and disorders responsive to the inhibition of Syk activity, which comprises administering to such patients an amount of at least one chemical entity effective to reduce signs or symptoms of the disease or disorder are provided. Also provided are methods for determining the presence or absence of Syk kinase in a sample.

Claims

1. At least one chemical entity chosen from compounds of Formula I: ##STR00097## and pharmaceutically acceptable salts thereof, wherein R.sub.1 is pyridinyl or pyrazolyl, each of which is optionally substituted, or R.sub.1 is ##STR00098## wherein A is an optionally substituted heteroaryl group having from 5 to 7 ring atoms including the atoms shared with the 6 membered aromatic ring; R.sub.2 is chosen from substituted aryl and optionally substituted heteroaryl; R.sub.3 is hydrogen; R.sub.4 is hydrogen; and R.sub.5 is hydrogen, provided that if R.sub.1 is 2-methoxy-pyridin-5-yl, then R.sub.2 is not 2,6-dimethylphenyl, 2-methoxyphenyl, 2-chlorophenyl, or 2-fluorophenyl; if R.sub.1 is indol-5-yl, then R.sub.2 is not 2-chlorophenyl, furan-2-yl, or 3-chloro-4-fluorophenyl; if R.sub.1 is 1H-indazol-5-yl, 1H-indol-6-yl, benzo[d]oxazole-6-yl, benzo[d]isoxazole-6-yl, benzothiazol-6-yl, or 3H-benzoimidazol-5-yl, then R.sub.2 is not 3-aminophenyl; and if R.sub.1 is 1H-indazol-6-yl, then R.sub.2 is not 3-carboxyphenyl or 4-carboxyphenyl.

2. At least one chemical entity of claim 1, wherein R.sub.1 is pyridinyl substituted with one or more groups chosen from hydroxy, optionally substituted amino, optionally substituted heterocycloalkyl, and lower alkoxy or pyrazolyl substituted with one or two groups chosen from cycloalkyl and lower alkyl substituted with one or more groups chosen from hydroxy and lower alkoxy.

3. At least one chemical entity of claim 2, wherein R.sub.1 is pyridinyl substituted with one or more groups chosen from hydroxy, heterocycloalkyl optionally substituted with one or two groups chosen from hydroxy, lower alkoxy, and lower alkyl, and amino optionally substituted with one or two groups chosen from lower alkyl, lower alkyl substituted with hydroxy, and lower alkyl substituted with lower alkoxy.

4. At least one chemical entity of claim 3, wherein R.sub.1 is chosen from 6-morpholinopyridin-3-yl, ((2-methoxyethyl)(methyl)amino)pyridin-3-yl, ((2-hydroxyethyl)(methyl)amino)pyridin-3-yl, 2-methoxypyridine-4-yl, (2-hydroxyethyl)-1H-pyrazol-4-yl, (2-hydroxypropyl)-1H-pyrazol-4-yl, (2-methoxyethyl)-1H-pyrazol-4-yl, 1-ethyl-1H-pyrazol-4-yl, 1-isopropyl-1H-pyrazol-4-yl, and 3-cyclopropyl-1H-pyrazol-5-yl.

5. At least one chemical entity of claim 1, wherein R.sub.1 is pyridinyl substituted with one or more groups chosen from hydroxy and lower alkoxy or pyrazolyl substituted with one or two groups chosen from cycloalkyl and lower alkyl substituted with one or more groups chosen from hydroxy and lower alkoxy.

6. At least one chemical entity of claim 5, wherein R.sub.1 is chosen from 2-methoxypyridine-4-yl, (2-hydroxyethyl)-1H-pyrazol-4-yl, (2-hydroxypropyl)-1H-pyrazol-4-yl, (2-methoxyethyl)-1H-pyrazol-4-yl, 1-ethyl-1H-pyrazol-4-yl, 1-isopropyl-1H-pyrazol-4-yl, and 3-cyclopropyl-1H-pyrazol-5-yl.

7. At least one chemical entity of claim 1, wherein R.sub.1 is ##STR00099##

8. At least one chemical entity of claim 7, wherein A is an optionally substituted pyrazolyl, oxazolyl, pyrrolyl, thiazolyl, or imidazolyl group.

9. At least one chemical entity of claim 8, wherein the imidazolyl group is substituted with lower alkyl.

10. At least one chemical entity of claim 7, wherein R.sub.1 is chosen from 1H-benzo[d]imidazol-6-yl, 1H-benzo[d]imidazol-5-yl, 1H-indazol-6-yl, 1H-indazol-5-yl, 1-methyl-1H-benzo[d]imidazol-6-yl, benzoxazol-6-yl, benzoxazol-5-yl, imidazo[1,2-a]pyridine-6-yl, 1H-indole-6-yl, 1H-indole-5-yl, benzothiazol-6-yl, and benzothiazol-5-yl.

11. At least one chemical entity of claim 1, wherein R.sub.2 is chosen from optionally substituted heteroaryl, dihydroindolyl, and dihydrobenzoxazinyl optionally substituted with oxo.

12. At least one chemical entity of claim 11, wherein R.sub.2 is chosen from 2,3-dimethyl-2H-indazol-6-yl, 1H-indazolyl-6-yl, 1-methyl-1H-indazol-5-yl, 1-methyl-1H-indazol-6-yl, 3,4-dihydro-2H-1,4-benzoxazin-3-one-6-yl, 1,3-benzoxazol-6-yl, 3-aminoquinolin-6-yl, and 2,3-dihydro-1H-indol-6-yl.

13. At least one chemical entity of claim 11, wherein R.sub.2 is chosen from 1H-indazolyl-6-yl, 1-methyl-1H-indazol-5-yl, 1-methyl-1H-indazol-6-yl, 3,4-dihydro-2H-1,4-benzoxazin-3-one-6-yl, 1,3-benzoxazol-6-yl, 3-aminoquinolin-6-yl, and 2,3-dihydro-1H-indol-6-yl.

14. At least one chemical entity according to claim 1, wherein the compound of Formula I is selected from: N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-1,3-benzodiazol-6-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indazol-6-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1-methyl-1H-1,3-benzodiazol-6-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1,3-benzoxazol-6-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1,3-benzoxazol-5-amine; 5-{[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]amino}pyridin-2-ol; N-{imidazo[1,2-a]pyridin-6-yl}-6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indazol-5-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indol-6-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indol-5-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1,3-benzothiazol-6-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1,3-benzothiazol-5-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-2-methoxypyridin-4-amine; 6-[8-(1H-1,3-benzodiazol-5-ylamino)imidazo[1,2-a]pyrazin-6-yl]-3,4-dihydro-2H-1,4-benzoxazin-3-one; 2-(4-{[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]amino}-1H-pyrazol-1-yl)ethan-1-ol; 3-(4-{[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]amino}-1H-pyrazol-1-yl)propan-1-ol; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1-(2-methoxyethyl)-1H-pyrazol-4-amine; 1-ethyl-N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-pyrazol-4-amine; N-[6-(1,3-benzoxazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-1,3-benzodiazol-6-amine; N-[6-(1H-1,3-benzodiazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indazol-6-amine; N-[6-(1-methyl-1H-indazol-5-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indazol-5-amine; N-[6-(3,4-dihydro-2H-1,4-benzoxazin-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-indazol-6-amine; N-[6-(3-aminoquinolin-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1,3-benzothiazol-5-amine; 6-{8-[(2-methoxypyridin-4-yl)amino]imidazo[1,2-a]pyrazin-6-yl}-3,4-dihydro-2H-1,4-benzoxazin-3-one; N-[6-(2,3-dihydro-1H-indol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-2-methoxypyridin-4-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1-(propan-2-yl)-1H-pyrazol-4-amine; 1-methyl-N-[6-(1-methyl-1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-1,3-benzodiazol-6-amine; 3-cyclopropyl-N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-1H-pyrazol-5-amine; N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-6-(morpholin-4-yl)pyridin-3-amine; N-[6-(2,3-dimethyl-2H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-6-(morpholin-4-yl)pyridin-3-amine; 5-N-[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]-2-N-(2-methoxyethyl)-2-N-methylpyridine-2,5-diamine; and 2-[(5-{[6-(1H-indazol-6-yl)imidazo[1,2-a]pyrazin-8-yl]amino}pyridin-2-yl)(methyl)amino]ethan-1-ol.

15. A pharmaceutical composition comprising at least one chemical entity of claim 1, together with at least one pharmaceutically acceptable vehicle chosen from carriers, adjuvants, and excipients.

16. A method for treating a patient having a disease responsive to the inhibition of Syk activity, comprising administering to the patient an effective amount of at least one chemical entity according to claim 1.

17. The method according to claim 16, wherein the patient is a human.

18. The method according to claim 16, wherein an effective amount of said at least one chemical entity is administered by a method chosen from intravenously, intramuscularly, and parenterally.

19. The method according to claim 16, wherein an effective amount of said at least one chemical entity is administered orally.

20. The method according to claim 16, wherein the disease responsive to inhibition of Syk activity is cancer.

21. The method according to claim 20, wherein the disease responsive to inhibition of Syk activity is B-cell lymphoma and leukemia.

22-32. (canceled)

33. A method for inhibiting B-cell activity comprising contacting cells expressing Syk with at least one chemical entity of claim 1, in an amount sufficient to detectably decrease B-cell activity in vitro.

34. (canceled)

Description

EXAMPLES

[0276] The invention is further illustrated by the following non-limiting examples.

[0277] In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

DME=dimethyl ether
DMEM=Dulbecco's modified Eagle's medium

DMF=N,N-dimethylformamide

[0278] DMSO=dimethylsulfoxide
Et.sub.2O=diethylether
g=gram
h=hour
mg=milligram
min=minutes
mL=milliliter
mmol=millimoles
mM=millimolar
ng=nanogram
nm=nanometer
nM=nanomolar
PBS=phosphate buffered saline
L=microliter
M=micromolar

Example I

Preparation of 6-(1H-indazol-6-yl)-N-(1-Isopropyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (5)

[0279] ##STR00007##

Preparation of 1-Isopropyl-4-nitro-1H-pyrazole (1)

[0280] Di-tert-butyl azodicarboxylate (1.59 g, 6.91 mmol) was added to a solution of 4-nitro-1H-pyrazole (601 mg, 5.32 mmol), 2-propanol (319 mg, 5.32 mmol) and triphenylphosphine (1.67 g, 6.36 mmol) in tetrahydrofuran (25 mL) over 3 minutes and the mixture was stirred at room temperature for 16 h. After this time, the reaction was concentrated under reduced pressure and the resulting residue purified by chromatography (silica, gradient, 1:4 ethyl acetate/heptane to 7:3 ethyl acetate/heptane) to afford 1-isopropyl-4-nitro-1H-pyrazole (1) (1.23 g, >100%) as an impure off-white solid, which was used without further purification: .sup.1H NMR (300 MHz, DMSO-d.sub.6) 8.91 (s, 1H), 8.24 (s, 1H), 4.58 (m, 1H), 1.42 (d, J=6.6 Hz, 6H).

Preparation of 1-isopropyl-1H-pyrazole-4-amine (2)

[0281] A suspension of impure 1-isopropyl-4-nitro-1H-pyrazole (1) (1.23 g), from above, in ethanol (50 mL) was treated with 10% palladium on carbon (400 mg, 50% water by weight) and shaken under a hydrogen atmosphere (35 psi) at room temperature for 1 h. After this time, the reaction was filtered through diatomaceous earth and the filtrate concentrated under reduced pressure to afford 1-isopropyl-1H-pyrazole-4-amine (2) (1.15 g) as an impure off-white solid, which was used without further purification: .sup.1H NMR (300 MHz, DMSO-d.sub.6) 7.02 (s, 1H), 6.87 (s, 1H), 4.26 (m, 1H), 3.72 (bs, 2H), 1.42 (d, J=6.6 Hz, 6H).

Preparation of 6,8-dibromoimidazo[1,2-a]pyrazine (3)

[0282] A 1-L four-neck round bottom flask equipped with a temperature probe, mechanical stirrer and reflux condenser was charged with 2-bromo-1,1-diethoxyethane (68.1 g, 346 mmol) and 48% aqueous hydrogen bromide (11.3 mL, 99.2 mmol), and the reaction mixture was stirred at reflux for 2 h. The resulting mixture was allowed to cool to 40 C. and solid sodium bicarbonate (8.50 g, 101 mmol) was added in small portions until gas evolution was observed to cease. Caution: initial addition of sodium bicarbonate to the warm solution resulted in vigorous gas evolution (foaming). The resulting suspension was filtered into a 1-L four-neck round bottomed flask and the filter cake was washed with ethanol (200 mL). The flask was equipped with a temperature probe, mechanical stirrer and reflux condenser. 3,5-Dibromopyrazin-2-amine (50.0 g, 198 mmol) was added and the reaction mixture was heated at reflux, with vigorous stirring, for 16 h. After this time, the suspension was cooled to 0 C. and filtered. The filter cake was washed with cold ethanol (50 mL), dried under vacuum and added to a 1-L three-neck round bottomed flask equipped with a mechanical stirrer. Water (200 mL) was added and the vigorously stirred suspension was treated portion-wise with solid potassium carbonate (27.4 g, 198 mmol). Caution: gas evolution upon the addition of potassium carbonate observed. After stirring for 30 min, the resulting precipitate was isolated by filtration and the filter cake washed with water (100 mL) followed by ethanol (50 mL). The filter cake was dried at 50 C. to a constant weight, under vacuum to provide 6,8-dibromoimidazo[1,2-a]pyrazine (3) (52.0 g, 94%) as a light yellow solid: .sup.1H NMR (300 MHz, DMSO-d.sub.6) 9.02 (s, 1H), 8.23 (s, 1H), 7.90 (s, 1H).

Preparation of 6-bromo-N-(1-isopropyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (4)

[0283] A mixture of impure 1-isopropyl-1H-pyrazole-4-amine (2) (1.00 g), from above, 6,8-dibromoimidazo[1,2-a]pyrazine (3) (750 mg, 2.71 mmol), and N,N-diisopropylethylamine (526 mg, 4.16 mmol) in DMF (20 mL) was stirred at 100 C. for 3 h. After this time, the reaction was cooled to room temperature and poured into ice water (200 mL). The resulting suspension was filtered and the filter cake dried to a constant weight under vacuum to afford impure 6-bromo-N-(1-isopropyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (4) (1.26 g) as an off-white solid, which was used without further purification: .sup.1H NMR (300 MHz, DMSO-d.sub.6) 10.26 (s, 1H), 8.14 (s, 1H), 8.08 (s, 1H), 7.90 (s, 1H), 7.77 (s, 1H), 7.59 (s, 1H), 4.49 (m, 1H), 1.40 (d, 6H); ESI MS m/z 323.3 [M+H].sup.+.

Preparation of 6-(1H-indazol-6-yl)-N-(1-Isopropyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (5)

[0284] A mixture of impure 6-bromo-N-(1-isopropyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (4) (195 mg), from above, and 1H-indazol-6-ylboronic acid (159 mg, 0.650 mmol) in 1 M aqueous sodium carbonate (1.3 mL) and 1,4-dioxane (3.5 mL) was sparged with nitrogen while stirring for 5 min. Tetrakis(triphenylphosphine)palladium(0) (69 mg, 0.060 mmol) was then added and the resulting mixture heated under microwave irradiation at 135 C. for 30 min. After this time, the reaction was diluted with methylene chloride (30 mL) and water (20 mL), and filtered through diatomaceous earth. The filtrate layers were separated and the aqueous phase was extracted with a mixture of 9:1 methylene chloride/methanol (100 mL). The combined organic layers were concentrated under reduced pressure and the resulting residue was purified by chromatography (silica, gradient, methylene chloride to 8:2 methylene chloride/methanol), then trituration with acetonitrile to afford 6-(1H-indazol-6-yl)-N-(1-isopropyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-amine (5) (85 mg, 35%) as a yellow solid: mp 250 C.; .sup.1H NMR (300 MHz, DMSO-d.sub.6) 13.23 (s, 1H), 9.95 (s, 1H), 8.59 (s, 1H), 8.24 (s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.97 (d, J=1.2 Hz, 1H), 7.85 (d, J=8.7 Hz, 1H), 7.75 (dd, J=8.7, 1.2 Hz, 1H), 7.62 (s, 1H), 4.52 (m, 1H), 1.47 (d, J=6.6 Hz, 6H); ESI MS m/z 359.4 [M+H]*; HPLC, 5.37 min, >99% (AUC).

Example 2

[0285] The following compounds were prepared using procedures similar to those described above. Those of ordinary skill in the art of organic synthesis will recognize when starting materials or reaction conditions should be varied to obtain the desired compound.

[0286] MS data reported in this example was obtained as follows: MS conditions: Electrospray MS is performed on a MICROMASS LCT equipped with a LockSpray source for accurate mass measurements. Spectra are acquired in positive ion mode from 100-1000 Da at an acquisition rate of 1 spectrum/0.9 s with a 0.1 s interscan delay. The instrument is tuned for a resolution of 5000 (FWHM). Every 5.sup.th scan is taken from the reference position of the Lockspray source. Leucine enkephalin (556.2771 [M+H].sup.+) is used as the reference, or lock mass.

TABLE-US-00001 Structure Name [M + H].sup.+ [00008] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1H-1,3- benzodiazol-6-amine 367.3 [00009] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1H-indazol-6-amine 367.3 [00010] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1-methyl-1H-1,3- benzodiazol-6-amine 381.3 [00011] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1,3-benzoxazol-6- amine 368.3 [00012] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1,3-benzoxazol-5- amine 368.3 [00013] 5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-ol 344.3 [00014] N-{imidazo[1,2-a]pyridin-6-yl}-6- (1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-amine 367.3 [00015] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1H-indazol-5-amine 367.3 [00016] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1H-indol-6-amine 366.3 [00017] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1H-indol-5-amine 366.3 [00018] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1,3-benzothiazol-6- amine 384.3 [00019] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1,3-benzothiazol-5- amine 384.3 [00020] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-2-methoxypyridin-4- amine 358.3 [00021] 6-[8-(1H-1,3-benzodiazol-5- ylamino)imidazo[1,2-a]pyrazin-6- yl]-3,4-dihydro-2H-1,4-benzoxazin- 3-one 398.1 [00022] 2-(4-{[6-(1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8- yl]amino}-1H-pyrazol-1-yl)ethan-1- ol 361.4 [00023] 3-(4-{[6-(1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8- yl]amino}-1H-pyrazol-1-yl)propan- 1-ol 375.2 [00024] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1-(2-methoxyethyl)- 1H-pyrazol-4-amine 375.4 [00025] 1-ethyl-N-[6-(1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-1H- pyrazol-4-amine 345 [00026] N-[6-(1,3-benzoxazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-1H- 1,3-benzodiazol-6-amine 368.2 [00027] N-[6-(1H-1,3-benzodiazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-1H- indazol-6-amine 367.1 [00028] N-[6-(1-methyl-1H-indazol-5- yl)imidazo[1,2-a]pyrazin-8-yl]-1H- indazol-5-amine 381.3 [00029] N-[6-(3,4-dihydro-2H-1,4- benzoxazin-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1H-indazol-6-amine 384.3 [00030] N-[6-(3-aminoquinolin-6- yl)imidazo[1,2-a]pyrazin-8-yl]-1,3- benzothiazol-5-amine 410.3 [00031] 6-{8-[(2-methoxypyridin-4- yl)amino]imidazo[1,2-a]pyrazin-6- yl}-3,4-dihydro-2H-1,4-benzoxazin- 3-one 389.7 [00032] N-[6-(2,3-dihydro-1H-indol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-2- methoxypyridin-4-amine 359.3 [00033] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-1-(propan-2-yl)-1H- pyrazol-4-amine 359.4 [00034] 1-methyl-N-[6-(1-methyl-1H- indazol-6-yl)imidazo[1,2-a]pyrazin- 8-yl]-1H-1,3-benzodiazol-6-amine 395.1 [00035] 3-cyclopropyl-N-[6-(1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-1H- pyrazol-5-amine 356.4 [00036] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-6-(morpholin-4-yl) pyridin-3-amine 413.2 [00037] N-[6-(2,3-dimethyl-2H-indazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 441.2 [00038] 5-N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-2-N-(2-methoxyethyl)- 2-N-methylpyridine-2,5-diamine 415.6 [00039] 1-[(6-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]amino}pyridin-3-yl)oxy]- 2-methylpropan-2-ol 415.6 [00040] 7-(8-{[1-(2-hydroxyethyl)-1H-pyrazol-4- yl]amino}imidazo[1,2-a]pyrazin-6-yl)- 1H,2H,3H-pyrido[2,3-b][1,4]oxazin-2- one 393.2 [00041] 2-(4-{[6-(3,4-dihydro-2H-1,4-benzoxazin- 7-yl)imidazo[1,2-a]pyrazin-8-yl]amino}- 1H-pyrazol-1-yl)ethan-1-ol 378.6 [00042] 6-(8-{[1-(2-hydroxyethyl)-1H-pyrazol-4- yl]amino}imidazo[1,2-a]pyrazin-6-yl)-3,4- dihydro-2H-1,4-benzoxazin-3-one 392.4 [00043] 2-(4-{[6-(1,3-benzothiazol-5- yl)imidazo[1,2-a]pyrazin-8-yl]amino}-1H- pyrazol-1-yl)ethan-1-ol 378.5 [00044] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-6-(2- methoxyethoxy)pyridin-3-amine 402.4 [00045] 6-(8-{[6-(morpholin-4-yl)pyridin-3- yl]amino}imidazo[1,2-a]pyrazin-6- yl)quinazolin-2-amine 440.3 [00046] 2-(4-{[6-(3,4-dihydro-2H-1,4-benzoxazin- 6-yl)imidazo[1,2-a]pyrazin-8-yl]amino}- 1H-pyrazol-1-yl)ethan-1-ol 378.6 [00047] 6-[2-(dimethylamino)ethoxy]-N-[6-(1H- indazol-6-yl)imidazo[1,2-a]pyrazin-8- yl]pyridin-3-amine 415.4 [00048] 1-(6-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]amino}pyridin-3-yl)-3- methylazetidin-3-ol 412.4 [00049] 2-[(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2- yl)oxy]ethan-1-ol 388.5 [00050] 3,3-dimethyl-6-(8-{[6-(morpholin-4- yl)pyridin-3-yl]amino}imidazo[1,2- a]pyrazin-6-yl)-2,3-dihydro-1H-indol-2- one 456.4 [00051] 1-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-3- methylazetidin-3-ol 413.4 [00052] 1-(6-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]amino}pyridin-3-yl)azetidin- 3-ol 398.1 [00053] 2-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-2- methylpropan-1-ol 400.2 [00054] 1-[(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)oxy]-2- methylpropan-2-ol 416.7 [00055] N-[5-(3,4-dihydro-2H-1,4-benzoxazin-6- yl)pyrazolo[1,5-a]pyrimidin-7-yl]-5- (morpholin-4-yl)pyridin-2-amine 429.47 [00056] N-[5-(1-methyl-1H-1,3-benzodiazol-6- yl)pyrazolo[1,5-a]pyrimidin-7-yl]-5- (morpholin-4-yl)pyridin-2-amine 427.1 [00057] 6-(7-{[5-(morpholin-4-yl)pyridin-2- yl]amino}pyrazolo[1,5-a]pyrimidin-5-yl)- 2,3-dihydro-1H-indol-2-one 428.1 [00058] N-[6-(3,4-dihydro-2H-1,4-benzoxazin-6- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 430.3 [00059] N-[6-(3,4-dihydro-2H-1,4-benzoxazin-7- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 430.3 [00060] 2-[(6-{[5-(1H-indazol-6-yl)pyrazolo[1,5- a]pyrimidin-7-yl]amino}pyridin-3- yl)(methyl)amino]ethan-1-ol 401.1 [00061] 6-(7-{[5-(morpholin-4-yl)pyridin-2- yl]amino}pyrazolo[1,5-a]pyrimidin-5-yl)- 3,4-dihydro-2H-1,4-benzoxazin-3-one 444.6 [00062] N-[5-(1H-indol-6-yl)pyrazolo[1,5- a]pyrimidin-7-yl]-5-(morpholin-4- yl)pyridin-2-amine 412.4 [00063] 6-(8-{[6-(morpholin-4-yl)pyridin-3- yl]amino}imidazo[1,2-a]pyrazin-6-yl)-2,3- dihydro-1H-indol-2-one 428.2 [00064] N-[6-(1-methyl-1H-1,3-benzodiazol-5- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 427 [00065] 2-[(5-{[6-(3,4-dihydro-2H-1,4-benzoxazin- 7-yl)imidazo[1,2-a]pyrazin-8-yl]amino} pyridin-2-yl)(methyl)amino]ethan-1-ol 418.6 [00066] N-[6-(1-methyl-1H-1,3-benzodiazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 427 [00067] 1-(5-{[6-(3,4-dihydro-2H-1,4-benzoxazin- 6-yl)imidazo[1,2-a]pyrazin-8- yl]amino}pyridin-2-yl)-4-methylpiperidin- 4-ol 458.2 [00068] N-[6-(1H-1,3-benzodiazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 413.3 [00069] 1-(5-{[6-(3,4-dihydro-2H-1,4-benzoxazin- 6-yl)imidazo[1,2-a]pyrazin-8- yl]amino}pyridin-2-yl)azetidin-3-ol 416.7 [00070] N-[5-(1H-1,3-benzodiazol-6- yl)pyrazolo[1,5-a]pyrimidin-7-yl]-5- (morpholin-4-yl)pyridin-2-amine 413.4 [00071] 1-(5-{[6-(1H-indol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-4- methylpiperidin-4-ol 440.3 [00072] N-[6-(1H-indol-6-yl)imidazo[1,2-a]pyrazin- 8-yl]-6-(morpholin-4-yl)pyridin-3-amine 412.2 [00073] 6-(8-{[6-(morpholin-4-yl)pyridin-3- yl]amino}imidazo[1,2-a]pyrazin-6-yl)-3,4- dihydro-2H-1,4-benzoxazin-3-one 444.8 [00074] 1-ethyl-N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]-5-methyl-1H-pyrazol-3- amine 358.2 [00075] 6-[8-({6-[(2- hydroxyethyl)(methyl)amino]pyridin-3- yl}amino)imidazo[1,2-a]pyrazin-6-yl]-3,4- dihydro-2H-1,4-benzoxazin-3-one 432.4 [00076] 1-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-3- methylpiperidin-3-ol 441.2 [00077] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]-6-(morpholin-4-yl)pyridazin- 3-amine 413.4 [00078] 7-(8-{[6-(morpholin-4-yl)pyridin-3- yl]amino}imidazo[1,2-a]pyrazin-6-yl)- 1H,2H,3H-pyrido[2,3-b][1,4]oxazin-2-one 445.5 [00079] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-6-[4-(propan-2- yl)piperazin-1-yl]pyridin-3-amine 454.1 [00080] N-[6-(4-fluoro-1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 431.4 [00081] 2-N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]-5-N-(2-methoxyethyl)-5-N- methylpyridine-2,5-diamine 414.2 [00082] 6-(1H-benzo[d]imidazol-6-yl)-N-(5- morpholinopyridin-2-yl)imidazo[1,2- b]pyridazin-8-amine 413.4 [00083] 6-(3-amino-1H-indazol-6-yl)-N-(5- morpholinopyridin-2-yl)imidazo[1,2- b]pyridazin-8-amine 428.1 [00084] 2-[(6-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]amino}pyridin-3- yl)(methyl)amino]ethan-1-ol 400.2 [00085] 1-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyridin-8-yl]amino}pyridin-2-yl)-4- methylpiperidin-4-ol 441.2 [00086] 1-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)azetidin- 3-ol 399.3 [00087] 6-(1H-indol-6-yl)-N-(5-morpholinopyridin- 2-yl)imidazo[1,2-b]pyridazin-8-amine 412.3 [00088] N-[6-(5-fluoro-1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8-yl]-6- (morpholin-4-yl)pyridin-3-amine 431.5 [00089] (3S)-1-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-3- methylpiperidin-3-ol 441.2 [00090] (3R)-1-(5-{[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-3- methylpiperidin-3-ol 441.2 [00091] 1-(5-{[6-(1H-indol-6-yl)imidazo[1,2- a]pyrazin-8-yl]amino}pyridin-2-yl)-3- methylazetidin-3-ol 412.4 [00092] [(2R)-4-(5-{[6-(1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8- yl]amino}pyridin-2-yl)morpholin-2- yl]methanol 443.5 [00093] [(2S)-4-(5-{[6-(1H-indazol-6- yl)imidazo[1,2-a]pyrazin-8- yl]amino}pyridin-2-yl)morpholin-2- yl]methanol 443.4 [00094] N-[6-(1H-indazol-6-yl)imidazo[1,2- a]pyrazin-8-yl]-2-(morpholin-4- yl)pyrimidin-5-amine 414.5 [00095] 1-ethyl-N-(6-{1H-pyrrolo[3,2-b]pyridin-6- yl}imidazo[1,2-a]pyrazin-8-yl)-1H-pyrazol- 4-amine 344.9 [00096] 2-{4-[(6-{1H-pyrrolo[3,2-b]pyridin-6- yl}imidazo[1,2-a]pyrazin-8-yl)amino]-1H- pyrazol-1-yl}ethan-1-ol 361.6

Example 3

Biochemical Syk Assay

[0287] A generalized procedure for one standard biochemical Syk Kinase Assay that can be used to test compounds disclosed in this application is as follows.

[0288] A master mix minus Syk enzyme is prepared containing 1 Cell Signaling kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na.sub.3VO.sub.4, 10 mM MgCl.sub.2), 0.5 M Promega PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, and 0.25% glycerol. A master mix plus Syk enzyme is prepared containing 1 Cell Signaling kinase buffer, 0.5 M PTK Biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton-X100, 0.25% glycerol and 0.4 ng/well Syk enzyme. Syk enzyme is purchased from Cell Signaling Technologies, expressed in baculovirus and is an N-terminally GST-tagged full length human wildtype Syk (accession number NM-00377). The Syk protein was purified in one step using glutathione-agarose. The purity of the final protein preparation was assessed by SDS-PAGE and Coomassie staining. A solution of 200 M ATP is prepared in water and adjusted to pH 7.4 with 1N NaOH. A quantity of 1.25 L of compounds in 5% DMSO is transferred to a 96-well area Costar polystyrene plate Compounds are tested singly and with an 11-point dose-responsive curve (starting concentration is 10-1 M; 1:2 dilution). A quantity of 18.75 L of master mix minus enzyme (as a negative control) and master mix plus enzyme is transferred to appropriate wells in 96-well area costar polystyrene plate. 5 L of 200 M ATP is added to that mixture in the 96-well area Costar polystyrene plate for final ATP concentration of 40 M. The reaction is allowed to incubate for 1 hour at room temperature. The reaction is stopped with Perkin Elmer 1 detection buffer containing 30 mM EDTA, 80 nM SA-APC, and 4 nM PT66 Ab. The plate is read using time-resolved fluorescence with a Perkin Elmer Envision using excitation filter 330 nm, emission filter 665 nm, and 2.sup.nd emission filter 615 nm. IC.sub.50 values are subsequently calculated using a linear regression algorithm.

Example 4

Ramos Cell pBLNK(Y96) Assay

[0289] Another generalized procedure for a standard cellular Syk Kinase Assay that can be used to test compounds disclosed in this application is as follows.

[0290] Ramos cells are serum starved at 210.sup.6 cells/ml in serum-free RPMI for 1 hour in an upright T175 Falcon TC flask. Cells are centrifuged (1100 rpm5 min) and incubated at a density of 0.510.sup.7 cells/ml in the presence of test compound or DMSO controls for 1 hr at 37 C. Cells are then stimulated by incubating with 10 g/ml anti-human IgM F(ab).sub.2 for 5 minutes at 37 C. Cells are pelleted, lysed in 40 ul cell lysis buffer, and mixed with Invitrogen SDS-PAGE loading buffer. 20 ul of cell lysate for each sample are subject to SDS-PAGE and western blotting with anti-phosphoBLNK(Tyr96) antibody (Cell Signaling Technology #3601) to assess Syk activity and anti-Syk antibody (BD Transduction Labs #611116) to control for total protein load in each lysate. The images are detected using fluorescent secondary detection systems and the LiCor Odyssey software.

Example 5

B-Cell Proliferation Assay

[0291] A generalized procedure for a standard cellular B-cell proliferation assay that can be used to test compounds disclosed in this application is as follows.

[0292] B-cells are purified from spleens of 8-16 week old Balb/c mice using a B-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Test compounds are diluted in 0.25% DMSO and incubated with 2.510.sup.5 purified mouse splenic B-cells for 30 min prior to addition of 10 g/ml of an anti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01) in a final volume of 100 l. Following 24 hr incubation, 1 Ci .sup.3H-thymidine is added and plates are incubated an additional 36 hr prior to harvest using the manufacturer's protocol for SPA[.sup.3H] thymidine uptake assay system (Amersham Biosciences # RPNQ 0130). SPA-bead based fluorescence is counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).

Example 6

T Cell Proliferation Assay

[0293] A generalized procedure for a standard T cell proliferation assay that can be used to test compounds disclosed in this application is as follows.

[0294] T cells are purified from spleens of 8-16 week old Balb/c mice using a Pan T cell isolation kit (Miltenyi Biotech, Cat #130-090-861). Test compounds are diluted in 0.25% DMSO and incubated with 2.510.sup.5 purified mouse splenic T cells in a final volume of 100 l in flat clear bottom plates precoated for 90 min at 37 C. with 10 g/ml each of anti-CD3 (BD #553057) and anti-CD28 (BD #553294) antibodies. Following 24 hr incubation, 1 Ci .sup.3H-thymidine is added and plates incubated an additional 36 hr prior to harvest using the manufacturer's protocol for SPA[.sup.3H] thymidine uptake assay system (Amersham Biosciences # RPNQ 0130). SPA-bead based fluorescence was counted in a microbeta counter (Wallace Triplex 1450, Perkin Elmer).

Example 7

CD69 Inhibition Assay

[0295] A generalized procedure for a standard assay for the inhibition of B cell activity that can be used to test compounds disclosed in this application is as follows.

[0296] Total mouse splenocytes are purified from spleens of 8-16 week old Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testing compounds are diluted to 0.5% DMSO and incubated with 1.2510.sup.6 splenocytes in a final volume of 200 l in flat clear bottom plates (Falcon 353072) for 60 min at 37 C. Cells are then stimulated with the addition of 15 g/ml IgM (Jackson ImmunoResearch 115-006-020), and incubated for 16 hr at 37 C., 5% CO.sub.2. Following the 16 hr incubation, cells are transferred to conical bottom clear 96-well plates and pelleted by centrifugation at 1200g5 min. Cells are preblocked by CD16/CD32 (BD Pharmingen #553142), followed by triple staining with CD19-FITC (BD Pharmingen #553785), CD69-PE (BD Pharmingen #553237), and 7AAD (BD Pharmingen #51-68981E). Cells are sorted on a BD FACSCalibur and gated on the CD19.sup.+/7AAD.sup. population. The levels of CD69 surface expression on the gated population is measured versus test compound concentration.

Example 8

BMMC Degranulation

[0297] A generalized procedure for a standard assay for bone-marrow derived mouse mast cell (BMMC) degranulation that can be used to test compounds disclosed in this application is as follows.

[0298] Bone-marrow derived mast cells were cultured for >4 weeks with IL-3 (10 ng/ml) and SCF (10 ng/ml). The cells were determined to be >90% cKit.sup.+/FceRI.sup.+ by FACS analysis at the time of use. Cells (610.sup.7 cells/50 ml) were serum-starved in a T150 tissue culture flask for 16 h in the absence of IL-3 and SCF containing IgE-DNP at 1 ug/ml. Overnight sensitized cells are washed twice in Tyrodes buffer and resuspended to 510.sup.6 cells/ml. 510.sup.5 cells (100 ul) are plated in a 96 well microtiter plate (Falcon 353072) and test compounds are serially diluted to a final concentration 0.25% DMSO in the plate for 1 hr at 37 C., 5% CO.sub.2. Wells are treated with a DNP-BSA antigen challenge (50 ng/ml) and incubated for and additional 30 min at 37 C. Supernatants are assayed for hexosamimidase release versus control wells. Cell pellets are simultaneously lysed and assessed for total hexosamimidase release to calculate specific release. Dose-response curves are generated using 4-parameter logistical fit and IC50s calculated.

Example 9

Passive Cutaneous Anaphylaxis (PCA)

[0299] The following is a procedure for a standard PCA model used for measuring in vivo IgE anti-DNP Ab sensitization and DNP-BSA antigen for triggering mast cell degranulation and release of immune regulators that cause acute vessel permeability monitored by Evan's blue dye into the inflamed area in the mouse ear.

[0300] Reagents: Anti-DNP IgE: is supplied as 1.2 mg/ml in a phosphate buffered solution with BSA for additional protein and azide for sterility. This is diluted 1:100 in sterile PBS as a 12 ug/ml working stock that can be further diluted in PBS to the appropriate concentration for injection. A further 1:5 dilution give a final 1:500 solution at 2.4 ng/ul. (10 ul/ear=24 ng). Sterile PBS alone will be used as a negative control. -DNP-BSA: will be made up at 4 mg/ml in sterile ddH.sub.2O and stored at 40 C. solution. It is further diluted 1:1 with sterile saline prior to use. This solution or a further dilution in saline is diluted 1:1 with 2% Evan's Blue in sterile saline that has been filtered though 0.02 um filter and refiltered prior to injection. For these experiments a final solution of 0.5 mg/ml of DNP-BSA in 1% Evans blue can be used. Tail vein injections will be held constant at 200 ul=100 ug in 1% Evan's Blue. -Evan's blue dye: A 2% stock in saline will be sterile filtered and diluted 1:1 with DNP-BSA saline solution for final concentration of 1% for injection.

General PCA Protocol Using Intradermal Ear Sensitization

[0301] 1) On d0, animals, anesthetized with isofluorine, are passively sensitized by intradermal injections of IgE anti-DNP using a 29-gauge insulin syringe. By convention, the right ear receives 10 ul intradermal injection of anti-DNP IgE while the left ear receives PBS. 2) 20 hr post sensitization, antigen challenge is administered by tail i.v. injection of DNP-BSA in 200 ul of 1% Evan's blue dye solution in saline. Tails are immersed in warm water prior to iv injection to improve success. 3) 30 minutes to 2 hr prior to this antigen challenge, drug is delivered sc or po in 10% EtOH/20% cremaphor/70% saline. 4) Animals are sacrifice by CO.sub.2 inhalation 30-60 min post antigen challenge and ears are removed for extraction of Evan's blue dye in 500 ul of formamide overnight at 65 C. 5) Blood is obtained by cardiac puncture just prior to final cervical dislocation and processed for plasma to provide PK analysis. 6) Evan's blue dye is quantified by reading absorbency of 200 ul of extracted solution in microtiter plates at 620 nm.

Study Design of Experiment

[0302] Each animal has one anti-DNP IgE sensitized ear (right ear by convention) and one PBS control ear (left ear by convention). Groups 1-8: represent the vehicle and compound testing arms; Group 9: represents the non-antigen negative control; Group 10: represents the non-sensitized challenged negative control; Group 11: represents the non-antigen challenged, non-sensitized negative control group (Groups 9-11 represent negative controls for background levels only and require only minimal number of animals per group.)

[0303] The compounds disclosed in the examples above were tested in the Syk biochemical assay described herein (Example 3) and certain of those compounds exhibited an IC.sub.50 value less than or equal to 1 micromolar. Certain of those compounds exhibited an IC.sub.50 value less than or equal to 100 nM. Certain of those compounds exhibited an IC.sub.50 value less than or equal to 10 nM. Certain of those compounds exhibited an IC.sub.50 value less than or equal to 1 nM.

[0304] Some of the compounds disclosed in Example 2 were tested in the B-cell proliferation assay (as described in Example 5) and exhibited an IC.sub.50 value less than or equal to 10 micromolar. Certain of those compounds exhibited an IC.sub.50 value less than or equal to 1 micromolar.

[0305] Certain of those compounds did not inhibit T-cell proliferation and had IC.sub.50 values greater than or equal to 5 micromolar when assayed under conditions described herein (as described in Example 6).

[0306] Certain compounds described herein exhibited IC.sub.50 values for inhibition of T-cell proliferation that were at least 3-fold, and in some instances 5-fold, greater than the IC.sub.50 values of those compounds for inhibition of B-cell proliferation.

[0307] Some of the compounds described herein were tested in an assay for inhibition of B cell activity (under the conditions described in example 7), and exhibited an IC.sub.50 value less than or equal to 10 micromolar. Certain of those compounds exhibited an IC.sub.50 value less than or equal to 1 micromolar.

[0308] Some of the compounds disclosed in described herein exhibited both biochemical and cell-based activity. For example, some of the compounds described herein exhibited an IC.sub.50 value less than or equal to 10 micromolar in the Syk biochemical assay described herein (Example 3) and an IC.sub.50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 4, 5, 7 or 8). Certain of those compounds exhibited an IC.sub.50 value less than or equal to 1 micromolar in the Syk biochemical assay described herein (Example 4) and an IC.sub.50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 4, 5, 7 or 8). Certain of those compounds exhibited an IC.sub.50 value less than or equal to 0.1 micromolar and an IC.sub.50 value less than or equal to 10 micromolar in at least one of the cell-based assays (other than the T-cell assay) described herein (Examples 4, 5, 7 or 8).

[0309] While some embodiments have been shown and described, various modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. For example, for claim construction purposes, it is not intended that the claims set forth hereinafter be construed in any way narrower than the literal language thereof, and it is thus not intended that exemplary embodiments from the specification be read into the claims. Accordingly, it is to be understood that the present invention has been described by way of illustration and not limitations on the scope of the claims.