NOVEL IMMUNE SYSTEM MODULATORS

Abstract

The present invention relates to a compound of Formula I:

##STR00001##

or a pharmaceutically acceptable salt thereof, wherein the symbols are as defined in the specification; a pharmaceutical composition comprising the same; and a method for treating or preventing autoimmunity disease using the same.

Claims

1. A method of inhibiting TLR-mediated immunostimulation in a mammalian species in need thereof, comprising administering to the mammalian species a therapeutically effective amount of at least one compound of at least one compound of Formula I or a pharmaceutically acceptable salt thereof, ##STR00319## wherein X is absent or is an optionally substituted alkyl, cycloalkyl, aryl, alkylaryl, or heterocycle; Q is H, (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, OR.sub.1, or SR.sub.1, in which q is 0 or 1; R.sub.1 and R.sub.2 are each independently hydrogen, alkyl, alkenyl, cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heterocycle, alkylheterocycle, or R.sub.1 and R.sub.2, when connected to N, together with the nitrogen atom to which they are bonded form a heterocycle, which may be optionally substituted by from one to four groups which may be the same or different selected from the group consisting of optionally substituted (C.sub.1-C.sub.4)alkyl, phenyl, benzyl, C(O)R.sub.12, (CH.sub.2).sub.pOR.sub.a, and (CH.sub.2).sub.pNR.sub.bR.sub.c; R.sub.7 is NR.sub.3R.sub.4; R.sub.3 and R.sub.4 are each independently hydrogen, alkyl, cycloalkyl, alkenyl, or alkylaryl, or R.sub.3 and R.sub.4 together with the nitrogen atom to which they are bonded form a heterocycle; Y is oxygen, sulfur, or NR.sub.11, where R.sub.11 is hydrogen, alkyl, cycloalkyl, alkenyl, or aryl group; R.sub.12 is alkyl, aryl, or heterocycle; L is alkyl or alkenyl containing from 2 to 10 carbon atoms; R.sub.5 is hydrogen, halogen, cyano, OR.sub.a, SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, NR.sub.bR.sub.c, S(O).sub.2NR.sub.bR.sub.c, C(O)OR.sub.a, C(O)R.sub.a, C(O)NR.sub.bR.sub.c, OC(O)R.sub.a, OC(O)NR.sub.bR.sub.c, NR.sub.bC(O)OR.sub.a, NR.sub.bC(O)R.sub.a, or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c; R.sub.6 is halogen, cyano, aryl, SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, NR.sub.bR.sub.c, S(O).sub.2NR.sub.bR.sub.c, C(O)OR.sub.a, C(O)R.sub.a, C(O)NR.sub.bR.sub.c, OC(O)R.sub.a, OC(O)NR.sub.bR.sub.c, NR.sub.bC(O)OR.sub.a, NR.sub.bC(O)R.sub.a, alkaryl, alkylheterocyclic, or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c; each occurrence of p is independently 2-4; each occurrence of R.sub.a is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; and each occurrence of R.sub.b and R.sub.c is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said R.sub.b and R.sub.c together with the nitrogen atom to which they are bonded optionally form a heterocycle comprising 1-4 heteroatoms, wherein the heterocycle is optionally substituted by (C.sub.1-C.sub.4)alkyl; provided that when R.sub.5 and R.sub.6 are H or methyl, then Q is not H.

2. The method of claim 1, wherein X is absent or is an aryl, or heterocycle; and Q is (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, OR.sub.1, or SR.sub.1, in which q is 0 or 1 and p is 2-4.

3. The method of claim 1, wherein X is absent or is aryl, or heterocycle; Q is (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, OR.sub.1, or SR.sub.1, in which q is 0 or 1 and p is 2-4; R.sub.1 and R.sub.2 are each independently hydrogen, alkyl, alkenyl, cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heterocycle, alkylheterocycle, or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are bonded form a heterocycle, which may be optionally substituted by from one to four groups which may be the same or different selected from (C.sub.1-C.sub.4)alkyl; R.sub.5 is hydrogen, halogen, cyano or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c; and R.sub.6 is halogen, cyano or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c.

4. The method of claim 1, wherein X is absent, alkyl or cycloalkyl and Q is (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, or SR.sub.1.

5. The method of claim 1, wherein R.sub.6 is alkaryl, alkylheterocyclic, or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c.

6. The method of claim 1, wherein Q is NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c.

7. The method of claim 1, wherein X is optionally substituted alkyl, cycloalkyl, aryl, or heterocycle.

8. The method of claim 1, wherein X is optionally substituted heterocycle.

9. The method of claim 7, wherein X is halogen-substituted alkyl, halogen-substituted cycloalkyl, halogen-substituted aryl, or halogen-substituted heterocycle.

10. The method of claim 1, wherein X is optionally substituted piperazine.

11. The method of claim 1, wherein Y is oxygen or sulfur.

12. The method of claim 1, wherein Y is NR.sub.11.

13. The method of claim 1, wherein R.sub.11 is H.

14. The method of claim 1, wherein L is CH.sub.2CH.sub.2.

15. The method of claim 1, wherein NR.sub.1R.sub.2, NR.sub.3R.sub.4, and NR.sub.bR.sub.c are each independently a heterocycle selected from the group consisting of: ##STR00320## in which R.sub.d is H, Me, CF.sub.3, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, CH.sub.2CMe.sub.3, Ph, CH.sub.2Ph, C(O)R.sub.12, (CH.sub.2).sub.pOR.sub.a, (CH.sub.2).sub.pC(O)OR.sub.a, optionally substituted pyridine, C(O)NR.sub.bR.sub.c, and (CH.sub.2).sub.pNR.sub.bR.sub.c, wherein R.sub.12 is alkyl, phenyl, or heterocycle; R.sub.a, R.sub.b and R.sub.c are each independently hydrogen, or (C.sub.1-C.sub.4)alkyl, or R.sub.b and R.sub.c, together with the nitrogen atom to which they are attached, form a saturated or unsaturated heterocyclic ring containing from three to seven ring atoms, which ring may optionally contain another heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and may be optionally substituted by from one to four groups which may be the same or different selected from the group consisting of alkyl, phenyl and benzyl; and p is 0-4.

16. The method of claim 1, wherein the compound has the structure of: ##STR00321## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.11, p, R.sub.b and R.sub.c are as described in claim 31.

17. The method of claim 16, wherein L is CH.sub.2CH.sub.2.

18. The method of claim 16, wherein R.sub.11 is H.

19. The method of claim 16, wherein NR.sub.3R.sub.4, NR.sub.1R.sub.2, and NR.sub.bR.sub.c are each independently ##STR00322##

20. The method of claim 16, wherein q is 0.

21. The method of claim 16, wherein R.sub.5 is halogen.

22. The method of claim 21, wherein R.sub.5 is Cl.

23. The method of claim 16, wherein R.sub.b is H.

24. The method of claim 16, wherein p is 2.

25. The method of claim 16, wherein the compound has the structure of: ##STR00323## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.b and R.sub.c are as described in claim 46.

26. The method of claim 25, wherein NR.sub.3R.sub.4, NR.sub.1R.sub.2, and NR.sub.bR.sub.c are each independently ##STR00324##

27. The method of claim 25, wherein R.sub.5 is Cl.

28. The method of claim 25, wherein NR.sub.1R.sub.2 is NHBu.

29. A method of inhibiting TLR-mediated immunostimulatory signaling, comprising contacting a cell expressing a TLR with an effective amount of at least one compound of Formula I or a pharmaceutically acceptable salt thereof, ##STR00325## wherein X is absent or is an optionally substituted alkyl, cycloalkyl, aryl, alkylaryl, or heterocycle; Q is H, (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, OR.sub.1, or SR.sub.1, in which q is 0 or 1; R.sub.1 and R.sub.2 are each independently hydrogen, alkyl, alkenyl, cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heterocycle, alkylheterocycle, or R.sub.1 and R.sub.2, when connected to N, together with the nitrogen atom to which they are bonded form a heterocycle, which may be optionally substituted by from one to four groups which may be the same or different selected from the group consisting of optionally substituted (C.sub.1-C.sub.4)alkyl, phenyl, benzyl, C(O)R.sub.12, (CH.sub.2).sub.pOR.sub.a, and (CH.sub.2).sub.pNR.sub.bR.sub.c; R.sub.7 is NR.sub.3R.sub.4; R.sub.3 and R.sub.4 are each independently hydrogen, alkyl, cycloalkyl, alkenyl, or alkylaryl, or R.sub.3 and R.sub.4 together with the nitrogen atom to which they are bonded form a heterocycle; Y is oxygen, sulfur, or NR.sub.11, where R.sub.11 is hydrogen, alkyl, cycloalkyl, alkenyl, or aryl group; R.sub.12 is alkyl, aryl, or heterocycle; L is alkyl or alkenyl containing from 2 to 10 carbon atoms; R.sub.5 is hydrogen, halogen, cyano, OR.sub.a, SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, NR.sub.bR.sub.c, S(O).sub.2NR.sub.bR.sub.c, C(O)OR.sub.a, C(O)R.sub.a, C(O)NR.sub.bR.sub.c, OC(O)R.sub.a, OC(O)NR.sub.bR.sub.c, NR.sub.bC(O)OR.sub.a, NR.sub.bC(O)R.sub.a, or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c; R.sub.6 is halogen, cyano, aryl, SR.sub.a, S(O)R.sub.a, S(O).sub.2R.sub.a, NR.sub.bR.sub.c, S(O).sub.2NR.sub.bR.sub.c, C(O)OR.sub.a, C(O)R.sub.a, C(O)NR.sub.bR.sub.c, OC(O)R.sub.a, OC(O)NR.sub.bR.sub.c, NR.sub.bC(O)OR.sub.a, NR.sub.bC(O)R.sub.a, alkaryl, alkylheterocyclic, or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c; each occurrence of p is independently 2-4; each occurrence of R.sub.a is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; and each occurrence of R.sub.b and R.sub.c is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said R.sub.b and R.sub.c together with the nitrogen atom to which they are bonded optionally form a heterocycle comprising 1-4 heteroatoms, wherein the heterocycle is optionally substituted by (C.sub.1-C.sub.4)alkyl; provided that when R.sub.5 and R.sub.6 are H or methyl, then Q is not H.

30. The method of claim 29, wherein X is absent or is an aryl, or heterocycle; and Q is (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, OR.sub.1, or SR.sub.1, in which q is 0 or 1 and p is 2-4.

31. The method of claim 29, wherein X is absent or is aryl, or heterocycle; Q is (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, OR.sub.1, or SR.sub.1, in which q is 0 or 1 and p is 2-4; R.sub.1 and R.sub.2 are each independently hydrogen, alkyl, alkenyl, cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heterocycle, alkylheterocycle, or R.sub.1 and R.sub.2 together with the nitrogen atom to which they are bonded form a heterocycle, which may be optionally substituted by from one to four groups which may be the same or different selected from (C.sub.1-C.sub.4)alkyl; R.sub.5 is hydrogen, halogen, cyano or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c; and R.sub.6 is halogen, cyano or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c.

32. The method of claim 29, wherein X is absent, alkyl or cycloalkyl and Q is (CH.sub.2).sub.qNR.sub.1R.sub.2, NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c, or SR.sub.1.

33. The method of claim 29, wherein R.sub.6 is alkaryl, alkylheterocyclic, or NR.sub.b(CH.sub.2).sub.pNR.sub.bR.sub.c.

34. The method of claim 29, wherein Q is NR.sub.1(CH.sub.2).sub.pNR.sub.bR.sub.c.

35. The method of claim 29, wherein X is optionally substituted alkyl, cycloalkyl, aryl, or heterocycle.

36. The method of claim 29, wherein X is optionally substituted heterocycle.

37. The method of claim 35, wherein X is halogen-substituted alkyl, halogen-substituted cycloalkyl, halogen-substituted aryl, or halogen-substituted heterocycle.

38. The method of claim 29, wherein X is optionally substituted piperazine.

39. The method of claim 29, wherein Y is oxygen or sulfur.

40. The method of claim 29, wherein Y is NR.sub.11.

41. The method of claim 29, wherein R.sub.11 is H.

42. The method of claim 29, wherein L is CH.sub.2CH.sub.2.

43. The method of claim 29, wherein NR.sub.1R.sub.2, NR.sub.3R.sub.4, and NR.sub.bR.sub.c are each independently a heterocycle selected from the group consisting of: ##STR00326## in which R.sub.d is H, Me, CF.sub.3, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, CH.sub.2CMe.sub.3, Ph, CH.sub.2Ph, C(O)R.sub.12, (CH.sub.2).sub.pOR.sub.a, (CH.sub.2).sub.pC(O)OR.sub.a, optionally substituted pyridine, C(O)NR.sub.bR.sub.c, and (CH.sub.2).sub.pNR.sub.bR.sub.c, wherein R.sub.12 is alkyl, phenyl, or heterocycle; R.sub.a, R.sub.b and R.sub.c are each independently hydrogen, or (C.sub.1-C.sub.4)alkyl, or R.sub.b and R.sub.c, together with the nitrogen atom to which they are attached, form a saturated or unsaturated heterocyclic ring containing from three to seven ring atoms, which ring may optionally contain another heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and may be optionally substituted by from one to four groups which may be the same or different selected from the group consisting of alkyl, phenyl and benzyl; and p is 0-4.

44. The method of claim 29, wherein the compound has the structure of: ##STR00327## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.11, p, R.sub.b and R.sub.c are as described in claim 59.

45. The method of claim 44, wherein L is CH.sub.2CH.sub.2.

46. The method of claim 44, wherein R.sub.11 is H.

47. The method of claim 44, wherein NR.sub.3R.sub.4, NR.sub.1R.sub.2, and NR.sub.bR.sub.c are each independently ##STR00328##

48. The method of claim 44, wherein q is 0.

49. The method of claim 44, wherein R.sub.5 is halogen.

50. The method of claim 49, wherein R.sub.5 is Cl.

51. The method of claim 44, wherein R.sub.b is H.

52. The method of claim 44, wherein p is 2.

53. The method of claim 44, wherein the compound has the structure of: ##STR00329## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.b and R.sub.c are as described in claim 74.

54. The method of claim 53, wherein NR.sub.3R.sub.4, NR.sub.1R.sub.2, and NR.sub.bR.sub.c are each independently ##STR00330##

55. The method of claim 53, wherein R.sub.5 is Cl.

56. The method of claim 53, wherein NR.sub.1R.sub.2 is NHBu.

Description

EXAMPLES

Example 1

[0788] ##STR00266##

[0789] A slurry of 3-amino-2-pyrazinecarboxylic acid (15 gm, 0.108 moles) in dry methanol (250 mL) was stirred as concentrated sulfuric acid (10 mL, 18.4 gm, 0.188 moles) was added. The addition of the acid caused most of the solid to dissolve. The mixture was stirred at reflux, causing the formation of a clear yellow solution. This solution was stirred at reflux for 5 hours and was then stored at room temperature overnight. The solution was diluted with methylene chloride (500 mL) and was stirred as a solution of potassium carbonate (26 gm, 0.188 moles) in water (75 mL) was slowly added. After stirring for 15 minutes, the organic phase was separated from the aqueous phase and was dried over magnesium sulfate. After filtration to remove the drying agent, the solvents were removed under reduced pressure. The solid residue was recrystallized from isopropyl alcohol to provide the methyl ester as a tan powder in a yield of 7.22 gm (43.7%).

##STR00267##

[0790] 2-Aminopyrazinecarboxylic acid methyl ester (2.2 gm, 0.0144 moles) and 4-bromobenzoyl chloride (11.0 gm, 0.05 moles) were combined in chloroform (50 mL) and pyridine (8 mL) was added. This mixture was stirred at 50 C. overnight. TLC (silica, 10% methanol in methylene chloride) showed that an appreciable amount of the 2-aminopyrazinecarboxylic acid methyl ester was left. Additional portions of 4-bromo-benzoyl chloride (5.5.0 gm, 0.025 moles) and pyridine (4 mL) were added and heating was continued at 65 C. overnight. TLC showed that all of the 2-aminopyrazinecarboxylic acid methyl ester had been consumed. The solution was cooled and methanol (25 mL) was added. After stirring for 30 minutes, the solvents were removed under reduced pressure. The solid residue was recrystallized from n-butanol.

##STR00268##

[0791] The solid methyl ester (from above) was suspended in a mixture of methanol (50 mL) and tetrahydrofuran (50 mL). This mixture was heated to boiling to provide a clear pale orange solution. The heat was removed and concentrated aqueous ammonia (25 mL) was slowly added. This solution was stirred without heating and within a few minutes, a solid began to separate. After stirring for 2 hours the mixture had cooled to room temperature and TLC (silica, 10% methanol in methylene chloride) showed formation of a new product spot. The mixture was filtered and the solid was washed with ether and dried. Yield=4.4 gm (95%) from 2-aminopyrazine-3-carboxylic acid methyl ester.

##STR00269##

[0792] To a solution of potassium hydroxide (3.8 gm, 0.058 moles) in water (60 mL) and DMSO (20 mL) was added the benzamide (2.18 gm, 6.810.sup.3 moles). This mixture was warmed slightly to help dissolution of the solid benzamide after which the clear yellow solution was stirred at room temperature for 45 minutes. An aliquot of the reaction solution was acidified with acetic acid and examined by TLC (silica, 10% methanol in methylene chloride). The starting material (rf=0.55) had been cleanly converted to a single new product (rf=0.43). The reaction was acidified to a pH of about 5.0 with acetic acid which caused the precipitation of product. Ice (50 gm) was added and the slurry was stirred until the ice had melted. The solid product was isolated by filtration and was washed well with water. After drying the yield was 2.06 gm (100%)

##STR00270##

[0793] A suspension of the pteridinone (2.06 gm, 6.810.sup.3 moles) in chloroform (50 mL) was stirred as thionyl chloride (4.05 gm, 2.48 mL, 0.034 moles) and DMF (1.0 mL) were added. This mixture was stirred at reflux for 1 hour. The clear yellow solution which had formed was examined by TLC (silica, 10% methanol in methylene chloride). The starting material (rf=0.43) had been cleanly converted to a single new product (rf=0.87). After cooling, the solvents were stripped under vacuum and the solid yellow residue was triturated in diethyl ether. This material was used for the next step without further purification.

##STR00271##

[0794] The chloropteridine from above was suspended in n-butanol (25 mL) and N-2-aminoethylmorpholine (1.77 gm, 1.78 mL, 1.3610.sup.2 moles) was added. This mixture was then heated at reflux for 30 minutes. After cooling, the butanol was evaporated under vacuum to give a pale yellow solid. This was partitioned between ethyl acetate (200 mL) and 5% sodium bicarbonate solution (200 mL). The ethyl acetate layer was isolated and was washed with 5% sodium bicarbonate solution (100 mL). Then the ethyl acetate solution was extracted with 5% HCl solution (250 mL). The combined acidic washes were backwashed with ethyl acetate (100 mL) and were then made basic by the addition of solid potassium carbonate. The precipitated solid was extracted into methylene chloride (2100 mL) and the combined extracts were dried over magnesium sulfate. After filtration to remove the magnesium sulfate, the methylene chloride was evaporated under reduced pressure to give the product as a tan solid. This was purified by chromatography on silica using 5% methanol in methylene chloride as eluent. The fraction containing the product gave a tan solid that weighed 1.6 gm (57% from the pteridinone) after evaporation of the solvents. The NMR of this fraction confirmed that it was the desired compound. It can be recrystallized from toluene.

[0795] A second run started with 2.16 gm (7.1310.sup.3 moles) of the hydroxyl compound. Chlorination was run as before with thionyl chloride/DMF in chloroform. Work-up: After cooling, the reaction solution was diluted with methylene chloride (100 mL) and this solution was washed with 10% sodium bicarbonate solution (200 mL). After drying (MgSO4) the solution was filtered and the solvents were evaporated under vacuum. The residual solid was dissolved in chloroform (100 mL) and N-2-amino-ethylmorpholine (1.89 gm, 1.9 mL, 1.4510.sup.2 moles) was added. This mixture was then heated at reflux for 30 minutes. After cooling, methylene chloride (200 mL) was added and this solution was washed with 5% sodium bicarbonate solution (200 mL). The methylene chloride layer was isolated and was extracted with 5% HCl solution (250 mL). The combined acidic washes were backwashed with methylene chloride (100 mL) and were then made basic by the addition of solid potassium carbonate. The precipitated solid was extracted into methylene chloride (2100 mL) and the combined extracts were dried over magnesium sulfate. After filtration to remove the magnesium sulfate, the methylene chloride was evaporated under reduced pressure to a volume of about 5 mL. Ethyl ether (100 mL) was added and the product quickly crystallized. The solid was isolated by filtration, washed with ether and dried. The yield was 1.87 gm (63%) from the hydroxyl compound.

##STR00272##

[0796] A 250 mL round bottom flask, equipped with a stir bar was dried in an oven and then cooled under nitrogen. To the cooled flask was added tris(dibenzylideneacetone)dipalladium (0) (22.8 mg, 2.510.sup.5 moles), +/binap (46.6 mg, 7.510.sup.5 moles), sodium t-butoxide (0.675 gm, 710.sup.3 moles) and toluene (25 mL). The flask was again flushed with nitrogen and the bromopteridine (1.81 gm, 4.3610.sup.3 moles) and N-methylpiperazine (0.600 gm, 6.010.sup.3 moles) were added. This mixture was stirred at 90 C. overnight. After cooling the reaction mixture was poured into a separatory funnel containing water (100 mL) and methylene chloride (100 mL). The organic extracts were washed with water and were then dried over magnesium sulfate. After filtration to remove the drying agent, the organic solvents were removed under vacuum. The residue was purified by chromatography on silica gel using 15% methanol in methylene chloride as eluent. The fractions containing the product were pooled and evaporated to give the product (Example 1) as an orange solid. Yield was 155 mg (8.2%). .sup.1H NMR: 2.81 ppm, singlet, 3H; 3.25 ppm, triplet, 6H; 3.6 ppm, multiplet, 8H; 4.1 ppm, multiplet, 2H; 4.25 ppm, triplet, 2H; 4.6, multiplet, 2H; 7.1 ppm, doublet, 2H; 8.1 ppm, doublet, 2H; 8.8 ppm, singlet, 1H; 8.9 ppm, singlet, 1H. LC/MS: M+1=435.35.

[0797] Hydrochloride salt formation: the pteridine (43 mg, 110.sup.4 moles) was dissolved in boiling ethanol. To this yellow solution was added concentrated hydrochloric acid (30 L). The solution was cooled which caused the tris-hydrochloride salt to crystallize as an orange solid. This was isolated by filtration and was washed with ethanol followed by diethyl ether. The solid salt was dried under vacuum. Yield=22.5 mg, Mw=543.91.

Examples 54 and 55

[0798] ##STR00273##

[0799] Methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate, (3.2 gm, 0.0144 moles) and benzoyl chloride (7.03 gm, 0.05 moles) are combined in chloroform (50 mL) and pyridine (8 mL) is added. This mixture is stirred at 50 C. overnight. Additional portions of benzoyl chloride (3.5.0 gm, 0.025 moles) and pyridine (4 mL) are added and heating is continued at 65 C. overnight. The solution is cooled and methanol (25 mL) is added. After stirring for 30 minutes, the solvents are removed under reduced pressure. The solid residue is recrystallized from n-butanol.

##STR00274##

[0800] A suspension of the dichlorocompound (2.6 gm, 8.010.sup.3 moles) in 2-propanol (25 mL) is stirred and treated with N-methylpiperazine (4.0 gm, 4.43 mL, 0.04 moles). This mixture is heated at reflux for one hour. Upon cooling on ice, the product can be crystallized and is isolated by filtration.

##STR00275##

[0801] The solid methyl ester (2.96 gm, 7.610.sup.3 moles) is suspended in a mixture of methanol (50 mL) and tetrahydrofuran (50 mL). This mixture is heated to boiling to provide a clear pale orange solution. The heat is removed and concentrated aqueous ammonia (25 mL) is slowly added. This solution is stirred without heating and within a few minutes, a solid begins to separate. After stirring for 2 hours the mixture is cooled to room temperature and TLC (silica, 10% methanol in methylene chloride) can be used to show the formation of a new product spot. The mixture is filtered and the solid is washed with ether and dried.

##STR00276##

[0802] To a solution of potassium hydroxide (4.0 gm, 0.06 moles) in water (60 mL) and DMSO (20 mL) is added the benzamide (2.7 gm, 7.210.sup.3 moles). This mixture is warmed slightly to help dissolution of the solid benzamide after which the clear yellow solution is stirred at room temperature for 45 minutes. An aliquot of the reaction solution is acidified with acetic acid and examined by TLC (silica, 10% methanol in methylene chloride). The reaction is acidified to a pH of about 5.0 with acetic acid which causes the precipitation of product. Ice (50 gm) is added and the slurry is stirred until the ice melts. The solid product is isolated by filtration and is washed well with water.

##STR00277##

[0803] A suspension of the pteridinone (2.06 gm, 6.810.sup.3 moles) in chloroform (50 mL) is stirred as thionyl chloride (4.05 gm, 2.48 mL, 0.034 moles) and DMF (1.0 mL) are added. This mixture is stirred at reflux for 1 hour After cooling, the chloroform solution is washed with saturated sodium bicarbonate solution and is then dried over magnesium sulfate. The chloroform solution is filtered and the solvents are stripped under vacuum to give a solid yellow residue which is triturated in diethyl ether. This material is used for the next step without further purification.

##STR00278##

[0804] The chlorinated pteridine from above is dissolved in chloroform (100 mL) and N-2-amino-ethylmorpholine (1.89 gm, 1.9 mL, 1.4510.sup.2 moles) is added. This mixture is then heated at reflux for 30 minutes. After cooling, methylene chloride (200 mL) is added and this solution is washed with 5% sodium bicarbonate solution (200 mL). The methylene chloride layer is isolated and is extracted with 5% HCl solution (250 mL). The combined acidic washes are backwashed with methylene chloride (100 mL) and are then made basic by the addition of solid potassium carbonate. The precipitated solid is extracted into methylene chloride (2100 mL) and the combined extracts are dried over magnesium sulfate. After filtration to remove the magnesium sulfate, the methylene chloride is evaporated under reduced pressure to a volume of about 5 mL. Ethyl ether (100 mL) is added and the product quickly crystallized. The solid is isolated by filtration, washed with ether and dried.

##STR00279##

[0805] A 250 mL Parr hydrogenation bottle is charged with chloride (468 mg, 1.010.sup.3 moles), ethanol (50 mL), sodium acetate (1.0 gm) and 10% palladium on carbon (500 mg). This mixture is hydrogenated at an initial hydrogen pressure of 50 PSI overnight. The Parr bottle is flushed with nitrogen and the contents were heated to boiling. The catalyst is removed by filtration of the hot mixture and the catalyst is washed with boiling ethanol (10 mL). The combined filtrates are concentrated under vacuum to about 10 mL and are then cooled on ice. The solid which separated is isolated by filtration and was dried under vacuum. LC/MS: M+1=435.4.

Example 56

[0806] ##STR00280##

[0807] The dichloropteridine (1.37 gm, 3.6410.sup.3 moles) and N-(2-aminoethyl)morpholine (1.0 gm, 7.6810.sup.3 moles) were added to 2-propanol (15 mL) and this mixture was heated at 60 C. Diisopropylethylamine (0.94 gm, 7.2810.sup.3 moles) was added and heating was continued overnight. TLC (silica, 10% methanol in methylene chloride) showed remaining dichloropteridine so additional N-(2-aminoethyl)morpholine (1.0 gm, 7.6810.sup.3 moles) was added. The temperature was increased to 85 C. for 2 hours and then the reaction was kept at room temperature overnight. The solvent was removed under reduced pressure and the remaining material was purified by chromatography on silica using 20% methanol in methylene chloride. The fractions containing the product were pooled and evaporated under reduced pressure. The remaining material was dissolved in ethanol (40 mL) and diethyl ether (100 mL). To this solution was added concentrated hydrochloric acid (500 L). The solid hydrochloride salt separated and was isolated by filtration. After washing with diethyl ether and drying there was obtained 400 mg of product as a yellow powder. .sup.1H NMR: 2.4 ppm, singlet, 3H, 2.7 ppm, doublet, 2H, 3.65 ppm, multiplet, 12H, 4.0 ppm, multiplet, 12H, 4.4 ppm, multiplet, 4H, 7.6 ppm, triplet, 2H, 7.65 ppm, quartet, 1H, 7.8 ppm, multiplet, 1H, 8.4 ppm, doublet, 2H, 9.5 ppm, broad singlet, 1H. LC/MS, M+2=564.5.

Example 57

[0808] ##STR00281##

[0809] The chloropteridine (1.76 gm, 4.9310.sup.3 moles) was stirred in THF (50 mL) to which was added a solution of ammonium formate (2.5 gm) dissolved in water (4 mL). To this was added 10% palladium on carbon (200 mg). This mixture was stirred under nitrogen at reflux for 30 minutes. TLC (silica, 25% methanol in methylene chloride) showed a single, blue fluorescent compound at Rf=0.43. The warm solution was filtered free of catalyst and the precipitated salts. The filter cake was washed with hot THF (250 mL) and the combined filtrates were washed with 1:1 brine and water. The solvents were removed under reduced pressure and the solid residue was stirred in diethyl ether (40 mL) before being isolated by filtration. After drying there was obtained 1.4 gm (88%) of the product as a tan solid.

##STR00282##

[0810] The hydroxypteridine (1.9 gm, 5.8910.sup.3 moles) was stirred in phosphorous oxychloride (25 mL) as diisopropylethylamine (761 mg, 5.8910.sup.3 mole) was added. This solution was heated at 75 C. for 6 hours and was then kept at room temperature overnight. Excess phosphorous oxychloride was removed under reduced pressure and the residual material was stirred with ice (30 gm) to destroy any remaining phosphorous oxychloride. This mixture was partitioned between 10% potassium carbonate solution (150 mL) and methylene chloride (150 mL). The methylene chloride solution was dried over magnesium sulfate before being filtered and evaporated under reduced pressure. The remaining material was used for the next step without further purification.

##STR00283##

[0811] The chloropteridine from above was dissolved in n-butanol (50 mL) and N-methyl-N-(2-aminoethyl)piperazine (2.0 gm, 1.410.sup.2 moles) was added. This mixture was heated at 110 C. for 30 minutes. TLC (silica, 25% methanol in methylene chloride) showed a single, blue fluorescent, product at Rf=0.093. The n-butanol was removed under reduced pressure and the residual material was extracted by stirring in diethyl ether (50 mL). This mixture was filtered and the solid filter cake was washed with diethyl ether (100 mL). The combined filtrates were extracted with water (50 mL). These aqueous extracts were treated with potassium carbonate to precipitate the product as an oil. The product was extracted into methylene chloride (100 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The remaining solid (1.28 gm) was dissolved in methanol (40 mL) and the solution was heated to reflux. Concentrated hydrochloric acid (982 L) was added and the solution was cooled on ice. The hydrochloride salt of Example 57 crystallized and was isolated by filtration. After being washed with methanol followed by diethyl ether, the solid was dried to give the product as its hydrochloride salt in a yield of 950 mg. LC/MS: M+1=448.45

Example 58

[0812] ##STR00284##

[0813] The chloropteridine (1.35 gm, 3.9610.sup.3 moles) was stirred in n-butanol (20 mL) and histamine (878 mg, 7.910.sup.3 moles) was added. This mixture was heated at 110 C. for 30 minutes. TLC (silica, 25% methanol in methylene chloride) showed a single, blue fluorescent, product at Rf=0.18. The reaction was diluted with diethyl ether (100 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (2150 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The remaining solid was stirred in diethyl ether (100 mL) and was then isolated by filtration to provide 850 mg of the product as a tan solid. LC/MS: M+1=416.27

Example 59

[0814] ##STR00285##

[0815] The chloropteridine (1.35 gm, 3.9610.sup.3 moles) was stirred in n-butanol (20 mL) and 4-(2-aminoethyl)pyridine (968 mg, 7.910.sup.3 moles) was added. This mixture was heated at 110 C. for 30 minutes. TLC (silica, 10% methanol in methylene chloride) showed a single, blue fluorescent, product at Rf=0.14. The reaction was diluted with diethyl ether (100 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (2150 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The remaining material was purified by chromatography on silica gel using 15% methanol in chloroform as eluent. The fractions containing the product were combined and evaporated to provide the product as a grey powder in a yield of 650 mg. LC/MS: M+1=427.26.

Example 60

[0816] ##STR00286##

[0817] The chloropteridine (852 mg, 2.510.sup.3 moles) was stirred in n-butanol (20 mL) and 2-(2-aminoethyl)pyridine (611 mg, 5.010.sup.3 moles) along with diisopropylethylamine (646 mg, 5.010.sup.3 moles) were added. This mixture was heated at 110 C. for 30 minutes. TLC (silica, 10% methanol in methylene chloride) showed a single, blue fluorescent, product at Rf=0.14. The reaction was diluted with diethyl ether (100 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (2150 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The residual oil crystallized on standing. This solid was stirred in diethyl ether (25 mL) and was then isolated by filtration to provide, after washing with diethyl ether and drying, 750 mg of the product as a tan solid. LC/MS: M+1=427.26.

Example 61

[0818] ##STR00287##

[0819] The chloropteridine (852 mg, 2.510.sup.3 moles) was stirred in n-butanol (20 mL) and S-(2-aminoethyl)isothiourea dihydrobromide (1.41 gm, 5.010.sup.3 moles) along with diisopropylethylamine (1.3 gm, 0.01 moles) were added. This mixture was heated at reflux for 30 minutes. The reaction was diluted with diethyl ether (200 mL) and this mixture was extracted with 5% hydrochloric acid (2100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The product separated as a pale yellow solid. This solid was isolated by filtration and washed with water to provide, after drying, 961 mg of the product as a yellow solid. LC/MS: M+1=424.21.

Example 62

[0820] ##STR00288##

[0821] The chloropteridine (852 mg, 2.510.sup.3 moles) was stirred in n-butanol (20 mL) and 2-(aminomethyl)-5-methylpyrazine (616 mg, 5.010.sup.3 moles) along with diisopropylethylamine (646 mg, 5.010.sup.3 moles) were added. This mixture was heated at 110 C. for 30 minutes. TLC (silica, 20% methanol in methylene chloride) showed a single, blue fluorescent, product at Rf=0.50. The reaction was diluted with diethyl ether (200 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (2150 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The residual oil crystallized upon being stirred in diethyl ether (150 mL) and was then isolated by filtration to provide, after washing with diethyl ether and drying, 600 mg of the product as a tan solid. LC/MS: M+1=428.23.

Example 63

[0822] ##STR00289##

[0823] The chloropteridine (500 mg, 1.4710.sup.3 moles) was stirred in n-butanol (20 mL) N-methyl-N-dimethylethylenediamine (300 mg, 2.9310.sup.3 moles) along with diisopropylethylamine (379 mg, 2.9310.sup.3 moles) were added. This mixture was heated at 110 C. for 30 minutes. TLC (silica, 20% methanol in methylene chloride) showed a single, blue fluorescent, product at Rf=0.14. The reaction was diluted with diethyl ether (200 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (3100 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The residual oil was dissolved in diethyl ether (100 mL) and to this solution was added a solution of sulfuric acid (144 mg, 1.4710.sup.3 moles) dissolved in diethyl ether (2.0 mL). The mixture containing the sulfate salt was stirred for one hour before the solid was isolated by filtration to provide, after washing with diethyl ether and drying, 425 mg of the product. LC/MS: M+1=407.5.

Example 64

[0824] ##STR00290##

[0825] The chloropteridine (852 mg, 2.510.sup.3 moles) was stirred in n-butanol (20 mL) and 3-[1,2,4]-triazol-1-yl-propylamine (631 mg, 5.010.sup.3 moles) along with diisopropylethylamine (646 mg, 5.010.sup.3 moles) were added. This mixture was heated at 110 C. for 8 hours. The reaction was diluted with diethyl ether (200 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (3100 mL). After drying over magnesium sulfate the methylene chloride solution was filtered and evaporated under reduced pressure. The residual oil crystallized upon being stirred in diethyl ether (100 mL) and was then isolated by filtration to provide, after washing with diethyl ether and drying, 350 mg of the product as a tan solid. LC/MS: M+1=431.35.

Example 65

[0826] ##STR00291##

[0827] The chloropteridine (852 mg, 2.510.sup.3 moles) was stirred in n-butanol (20 mL) and 2,4-diamino-6-(3-aminopropyl)amino-1,3,5-triazine (916 mg, 5.010.sup.3 moles) along with diisopropylethylamine (646 mg, 5.010.sup.3 moles) were added. This mixture was heated at 110 C. for 5 hours. The reaction was diluted with diethyl ether (200 mL) and this mixture was extracted with 5% hydrochloric acid (100 mL) followed by water (100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with n-butanol (2100 mL) and the combined extracts were washed with brine (50 mL). After drying over sodium sulfate the n-butanol solution was filtered and evaporated under reduced pressure to a volume of approximately 25 mL. This solution was cooled in the freezer overnight. The solid which crystallized was isolated by filtration to provide, after washing with n-butanol followed by diethyl ether and drying, 900 mg of the product as a tan solid. LC/MS: M+1=488.37.

Examples 66 and 67

[0828] ##STR00292##

[0829] A suspension of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (25.0 gm, 0.113 moles) in 2-propanol (200 mL) was stirred as pyrrolidine (8.84 gm, 0.124 moles) was added. To this mixture was added diisopropyethylamine (16.3 gm, 0.126 moles) after which the reaction was heated to reflux. At reflux, a brown solution resulted. After 2 hours at reflux, TLC (silica, 1:1 ethyl acetate and hexane) showed all of the starting material had been consumed with the formation of a single product. The reaction was cooled to room temperature which caused the product to crystallize. The solid product was isolated by filtration and was washed with 2-propanol and then with diethyl ether. After drying there was obtained 24.8 gm (85.5%) of the product as a pink solid.

##STR00293##

[0830] The pyrazine (24.8 gm, 9.6610.sup.2 moles) was stirred in pyridine (200 mL) as benzoyl chloride (33.9 gm, 0.241 moles) was added in 3 portions. This solution was stirred at 65 C. overnight. After cooling, the pyridine was removed under reduced pressure and the remaining material was dissolved in methylene chloride (400 mL) and water (200 mL) was added. To this mixture was added potassium carbonate until the aqueous was basic to litmus. The methylene chloride solution was isolated and washed with 2% HCl (250 mL.). The solution was then dried over magnesium sulfate. After filtration, the solvents were removed under reduced pressure. The remaining material was stirred with diethyl ether (200 mL) causing the product to crystallize. The solid product was isolated by filtration. After washing with diethyl ether and drying the imide was obtained as a grey solid in a yield of 36.8 gm (82%).

##STR00294##

[0831] The imide (28.0 gm, 0.06 moles) was stirred in THF in a 500 mL pressure flask. To this was added concentrated ammonia (40 mL). The flask was sealed and heated at 65 C. for 3 hours. After cooling, the solid which crystallized was isolated by filtration. This solid proved to be starting imide. The filtrates were shown by TLC (silica, 1:1 ethyl acetate and hexane) to contain more imide (Rf=0.56) along with another compound (Rf=0.44). After evaporation of the solvents under reduced pressure, these two materials were separated by chromatography on silica using 2.5% methanol in methylene chloride. The fractions containing the product were pooled and evaporated to give the product as a white solid in a yield of 1.9 gm. LC/MS: M+1=346.25.

##STR00295##

[0832] A slurry of the pyrazine (1.8 gm, 5.4910.sup.3 moles) in DMSO (25 mL) was stirred as a solution of potassium hydroxide (85%, 2.94 gm, 4.4510.sup.2 moles) in water (25 mL) was added. This mixture was warmed to 60 C. and was then stirred at room temperature for 30 minutes. The thick slurry was diluted with water (25 mL) and acetic acid (2.67 gm, 4.4510.sup.2 moles) was added. After stirring for 10 minutes, the pteridinone was isolated by filtration, washed with water and dried. The yield of pteridinone was 1.6 gm, (88.9%).

##STR00296##

[0833] The pteridinone (1.6 gm, 4.8810.sup.3 moles), phosphorous oxychloride (25 mL) and diisopropylethylamine (636 mg, 4.9210.sup.3 moles) were combined and heated at 80 C. for 8 hours. The excess phosphorous oxychloride was removed under reduced pressure and the remaining material was stirred with methylene chloride (100 mL). Ice and water (100 gm) were added with stirring and potassium carbonate was added until the pH of the aqueous was 7.0. The methylene chloride solution was isolated and dried over magnesium sulfate. After filtration, the methylene chloride was evaporated under reduced pressure. The remaining dichloropteridine was used in the next step without further purification.

##STR00297##

[0834] The dichloropteridine from above and N-(2-aminoethyl)morpholine (2.54 gm, 1.9510.sup.2 moles) were added to n-butanol (50 mL). This mixture was heated at 110 C. for 12 hours. TLC (silica, 10% methanol in methylene chloride) showed remaining dichloropteridine so additional N-(2-aminoethyl)morpholine (2.54 gm, 1.9510.sup.2 moles) was added. Heating at 110 C. was continued for an additional 12 hours. TLC (silica, 10% methanol in methylene chloride) showed two compounds at Rf=0.62 and at Rf=0.33. The solvent was removed under reduced pressure and the remaining material was purified by chromatography on silica using 10% methanol in methylene chloride and switching to 15% methanol in methylene chloride. The fractions containing the two compounds were pooled and evaporated under reduced pressure. Compound 1 with an Rf of 0.62 was identified as Example 66 and was isolated in a yield of 572 mg. Compound 2 with an Rf of 0.33 was identified as Example 67 and was isolated in a yield of 250 mg. LC/MS: Example 67, M+1=593.54. Example 66, M+1=534.45.

Example 68

[0835] ##STR00298##

[0836] A suspension of methyl 3-amino-5,6-dichloro-2-pyrazinecarboxylate (5.0 gm, 2.2510.sup.2 moles) in 2-propanol (50 mL) was stirred as N-methylpiperazine (2.48 gm, 2.4810.sup.2 moles) was added. To this mixture was added diisopropyethylamine (3.2 gm, 2.4810.sup.2 moles) after which the reaction was heated to reflux. At reflux, a brown solution resulted. After 2 hours at reflux, TLC (silica, 10% methanol in methylene chloride) showed all of the starting material had been consumed with the formation of a single product. The reaction was cooled to room temperature overnight which caused the product to crystallize. The solid product was isolated by filtration and was washed with 2-propanol and then with diethyl ether. After drying there was obtained 5.8 gm (90.2%) of the product as a pink solid.

##STR00299##

[0837] The pyrazine (9.5 gm, 3.3210.sup.2 moles) was stirred in pyridine (60 mL) as 4-t-butylbenzoyl chloride (16.5 gm, 8.410.sup.2 moles) was added in 3 portions. This solution was stirred at 65 C. for 20 hours. After cooling, the pyridine solution was poured into water (300 mL) and the precipitated solid was extracted into methylene chloride (2200 mL). The combined extracts were then dried over magnesium sulfate. After filtration, the solvents were removed under reduced pressure. The remaining material was stirred with diethyl ether (100 mL). The solid product was isolated by filtration. After washing with diethyl ether and drying the imide was obtained as a tan solid in a yield of 13.5 gm (67%).

##STR00300##

[0838] The imide (13.5 gm, 2.2310.sup.2 moles) was stirred in a mixture of THF (25 mL) and methanol (65 mL). To this was added concentrated ammonia (40 mL). The mixture was heated to reflux for 2 hours. After cooling, the solvents were removed under reduced pressure, and the remaining material was dissolved in methylene chloride (250 mL). The methylene chloride solution was washed with 5% potassium carbonate (100 mL) and then water (100 mL). After drying over magnesium chloride, the solution was filtered and evaporated under reduced pressure. The remaining material was stirred with diethyl ether (100 mL). The solid product which crystallized was isolated by filtration. After washing with diethyl ether and drying the product was obtained as a tan solid in a yield of 9.1 gm (94.7%).

##STR00301##

[0839] The chloropyrazine (15 gm, 3.4810.sup.2 moles) was dissolved in DMF (250 mL) and 10% palladium on carbon (200 mg) was added. This mixture was hydrogenated at 50 PSI on a Parr apparatus until hydrogen consumption stopped (2 hours). The resulting slurry was transferred to a flask and heated to boiling. The hot solution was filtered to remove catalyst and the filtrates were cooled in the freezer overnight. The solid which had crystallized from solution was isolated by filtration, washed with DMF followed by diethyl ether and dried. The hydrochloride salt of the product was isolated in a yield of 5.4 gm (36%). LC/MS: M+1=39735.

##STR00302##

[0840] A slurry of the pyrazine (1.8 gm, 5.4910.sup.3 moles) in DMSO (25 mL) was stirred as a solution of potassium hydroxide (85%, 2.94 gm, 4.4510.sup.2 moles) in water (25 mL) was added. This mixture was warmed to 60 C. and was then stirred at room temperature for 30 minutes. The thick slurry was diluted with water (25 mL) and acetic acid (2.67 gm, 4.4510.sup.2 moles) was added. After stirring for 10 minutes, the pteridinone was isolated by filtration, washed with water and dried. The yield of pteridinone was 1.6 gm, (88.9%).g

##STR00303##

[0841] A slurry of the pyrazine (4.3 gm, 1.010.sup.2 moles) in DMSO (45 mL) was stirred as a solution of potassium hydroxide (85%, 5.36 gm, 8.1110.sup.2 moles) in water (45 mL) was added. This mixture was warmed to 80 C. and was then stirred at that temperature for 2 hours. The thick slurry was diluted with water (50 mL) and acetic acid (4.86 gm, 8.1110.sup.2 moles) was added. After stirring for 10 minutes, the dihydroxypteridine was isolated by filtration, washed with water and dried. The yield of dihydroxypteridine was 2.96 gm, (75%). LC/MS: M+1=395.33.

##STR00304##

[0842] The dihydroxypteridine (2.86 gm, 7.2510.sup.3 moles), phosphorous oxychloride (50 mL), chloroform (50 mL) and diisopropylethylamine (1.87 gm, 1.4510.sup.2 moles) were combined and heated to reflux for 6 hours. The excess phosphorous oxychloride and chloroform were removed under reduced pressure and the remaining material was stirred with methylene chloride (200 mL). Ice and water (100 gm) were added with stirring and sodium bicarbonate was added until the pH of the aqueous was 8.0. The methylene chloride solution was isolated and dried over magnesium sulfate. After filtration, the methylene chloride was evaporated under reduced pressure. The remaining dichloropteridine was used in the next step without further purification.

##STR00305##

[0843] The dichloropteridine from above and N-(2-aminoethyl)morpholine (3.77 gm, 2.9010.sup.2 moles) were added to n-butanol (25 mL). This mixture was heated at 110 C. for 12 hours. The reaction was diluted with diethyl ether (200 mL) and this mixture was extracted with 5% hydrochloric acid (2100 mL). The combined aqueous extracts were washed with diethyl ether (100 mL) before being made basic by the addition of potassium carbonate. The basic aqueous mixture was extracted with methylene chloride (2150 mL) and the combined extracts were dried over magnesium sulfate. The methylene chloride solution was filtered and evaporated under reduced pressure. The remaining material was purified by chromatography on silica using 25% methanol in methylene chloride and switching to 25% methanol and 2% methylamine in methylene chloride. Unreacted dichloropteridine was eluted first and the product was eluted upon switching the eluent to 25% methanol and 2% methylamine in methylene chloride. The fractions containing the product were pooled and evaporated under reduced pressure to provide Example 68 in a yield of 250 mg. LC/MS: M+1=619.54.

Example 69

[0844] ##STR00306##

[0845] Benzamidine hydrochloride (26.0 gm, 0.166 moles) and dimethyl malonate (21.9 gm, 0.166 moles) were combined in dry methanol (200 mL). This mixture was stirred as 30% sodium methoxide in methanol (89.7 gm, 0.498 moles) was added. A precipitate of sodium chloride formed and this mixture was stirred at 55 C. for 2 hours. The reaction mixture was diluted with water (500 mL) to form a clear solution. This was acidified by the addition of acetic acid (35 mL) causing a white precipitate to quickly form. After stirring for 30 minutes, the solid was isolated by filtration. The filter cake was washed, in turn with water, methanol and acetone. After drying, the 2-phenyl-4,6-dihydroxypyrimidine was obtained in a yield of 25 gm (80%) as a white solid.

##STR00307##

[0846] Solution 1:

[0847] Aniline (9.3 gm, 0.10 moles) was dissolved in water (200 mL) and Ice (100 gm) containing concentrated hydrochloric acid (20 mL). This solution was stirred as a solution of sodium nitrite (6.9 gm, 0.10 moles) dissolved in water (50 mL) was dripped in. Once the addition was complete the diazonium solution was kept on ice as solution 2 was prepared.

[0848] Solution 2:

[0849] Sodium hydroxide (24 gm, 0.60 moles) and 2-phenyl-4,6-dihydroxypyrimidine (18.8 gm, 0.10 moles) were dissolved in water (200 mL) and once dissolution was complete, ice (100 gm) was added.

[0850] Solution 1 was slowly poured into solution 2 at ice temperature with stirring. The resulting bright orange solution was stirred and the sodium salt of the azo compound soon crystallized forming a thick slurry. After 30 minutes, the thick slurry was acidified with concentrated hydrochloric acid and after sitting for 30 minutes, the azo compound was isolated by filtration. The damp solid was washed with water and dried to provide the azopyrimidine as a yellow solid in a yield of 12.8 gm (43.7%)

##STR00308##

[0851] The dihydroxyphenylazopyrimidine (11.7 gm, 0.04 moles) was powdered and mixed with phosphorous oxychloride (45 mL). This mixture was stirred as diisopropylethylamine (12.3 mL) was slowly added. The resulting orange slurry was heated at reflux for 1 hour. Upon cooling, excess phosphorous oxychloride was removed under reduced pressure. The residual material was treated with ice and this was then extracted with methylene chloride (300 mL). The methylene chloride extracts were washed with water (2150 mL) before being dried over magnesium sulfate. After filtration, the solvent was removed under reduced pressure. The residual material was stirred with a 50/50 mixture of ethyl acetate and hexane (50 mL). The resulting solid was isolated by filtration and was washed with a 50/50 mixture of ethyl acetate and hexane. After drying, there was obtained 10.5 gm (79.7%) of the product as an orange solid.

##STR00309##

[0852] A mixture of 2-phenyl-4,6-dichloro-5-phenylazopyrimidine (6.58 gm, 2.010.sup.2 moles) and N-(2-aminoethyl)morpholine (10.4 gm, 8.010.sup.2 moles) in n-butanol (50 mL) was heated to boiling. The thick slurry which initially formed was transformed into a solution as the mixture reached reflux. After 1 hour at reflux the hot solution was diluted with 2-propanol (100 mL) keeping the solution at reflux. After the addition of 2-propanol, heating at reflux was continued for an additional 1 hour. Upon cooling, the product crystallized as a yellow solid. After cooling on ice the solid was isolated by filtration, washed with 2-propanol and dried to provide the product as a granular orange solid in a yield of 9.8 gm, (94.8%).

##STR00310##

[0853] The phenylazopyrimidine (16.5 gm, 3.1910.sup.2 moles) was stirred in methanol (100 mL) and THF (100 mL). To this was added 10% palladium on carbon (500 mg). The solution was heated to reflux and ammonium formate (16 gm) dissolved in water (25 mL) was added in 3 portions over a period of 1 hour. After being heated at reflux for 2 hours, the orange color of the azopyrimidine had faded and TLC (silica, 10% methanol in methylene chloride) showed consumption of the azo compound with a single product (Rf=0.13). After cooling, the catalyst was removed by filtration and the filtrates were evaporated under reduced pressure. The remaining material was partitioned between methylene chloride (400 mL) and 10% potassium carbonate solution (100 mL). The solution was dried over magnesium sulfate. After filtration to remove the magnesium sulfate, the solvents were removed under reduced pressure. To the remaining material was added diethyl ether (100 mL) and this mixture was stirred for 15 minutes. The solid was isolated by filtration and was washed with diethyl ether. After drying there was obtained the triaminopyrimidine as a tan solid in a yield of 11.2 gm, (82%).

##STR00311##

[0854] The triaminopyrimidine (0.855 gm, 2.010.sup.3 moles) and ethyl pyruvate (0.348 gm, 3.010.sup.3 moles) were combined in 2-butanol (10 mL). This mixture was heated to reflux forming a clear yellow solution. After being heated at reflux for 2 hours, TLC (silica, 15% methanol in methylene chloride) showed a single, blue fluorescent, product. The solution was cooled to room temperature causing the pteridinone to crystallize. To the slurry was added 2-propanol (10 mL) and stirring was continued for another 15 minutes. The pteridinone was isolated by filtration, washed with 2-propanol, and dried. The yield was 0.75 gm (78.2%). LCMS: M+1=480.3. .sup.1H NMR (CDCl.sub.3): 2.57 ppm, singlet, 3H, 2.63 ppm, multiplet, 8H, 2.76 ppm, triplet, 2H, 2.80 ppm, triplet, 2H, 3.65 ppm, triplet, 4H, 3.79 ppm, triplet, 4H, 3.86 ppm, quartet, 2H, 4.64 ppm, triplet, 2H, 6.99 ppm, triplet, 1H, 7.50 ppm, multiplet, 3H, 8.48 ppm, triplet, 2H.

Example 70

[0855] ##STR00312##

[0856] The triaminopyrimidine (0.855 gm, 2.010.sup.3 moles) and ethyl benzoylformate (0.535 gm, 3.010.sup.3 moles) were combined in 2-butanol (10 mL). This mixture was heated to 100 C. for 16 hours. The dark red solution was cooled to room temperature overnight causing the pteridinone to crystallize. The pteridinone was isolated by filtration, washed with 2-butanol followed by diethyl ether, and dried. The yield was 0.123 gm (11.4%). LC/MS: M+1=542.4.

[0857] .sup.1H NMR (CDCl.sub.3): 2.63 ppm, multiplet, 4H, 2.67 ppm, multiplet, 4H, 2.77 ppm, triplet, 2H, 2.85 ppm, triplet, 2H, 3.66 ppm, multiplet, 4H, 3.81 ppm, multiplet, 4H, 3.87 ppm, multiplet, 2H, 4.73 ppm, triplet, 2H, 7.31 ppm, multiplet, 2H, 7.74 ppm, multiplet, 6H, 8.39 ppm, triplet, 2H, 8.52 ppm, doublet, 2H.

Example 71

[0858] ##STR00313##

[0859] The triaminopyrimidine (0.855 gm, 2.010.sup.3 moles) was dissolved in warm 2-butanol (10 mL). To the warm solution was added ethyl trifluoromethylpyruvate (0.510 gm, 3.010.sup.3 moles). This mixture was capped with a septum and was stirred at room temperature overnight. The volatiles were removed under reduced pressure and the remaining material was dissolved in diethyl ether (25 mL). The solution was stirred for a few minutes causing the pteridinone to crystallize. The pteridinone was isolated by filtration, washed with diethyl ether, and dried. The yield was 0.750 gm (70.3%). LC/MS: M+1=534. .sup.1H NMR (CDCl.sub.3): 2.61 ppm, broad singlet, 8H, 2.76 ppm, triplet, 2H, 2.82 ppm, triplet, 2H, 3.61 ppm, multiplet, 4H, 3.78 ppm, multiplet, 4H, 3.86 ppm, multiplet, 2H, 4.66 ppm, triplet, 2H, 7.32 ppm, multiplet, 1H, 7.55 ppm, multiplet, 3H, 8.49 ppm, doublet, 2H.

Example 72

[0860] ##STR00314##

[0861] The triaminopyrimidine (0.855 gm, 2.010.sup.3 moles) and ethyl-4-cyanobenzoylformate (0.610 gm, 3.010.sup.3 moles) were combined in 2-butanol (10 mL). This mixture was heated to 95 C. forming a yellow solution. Heating at 95 C. was continued overnight. The resulting slurry was cooled to room temperature and diluted with diethyl ether (25 mL). After stirring for 15 minutes the pteridinone was isolated by filtration, washed with diethyl ether, and dried. The yield of the bright yellow product was 0.860 gm (76%). LC/MS: M+1=567. .sup.1H NMR (CDCl.sub.3): 2.64 ppm, multiplet, 8H, 2.79 ppm, triplet, 2H, 2.85 ppm, triplet, 2H, 3.65 ppm, triplet, 4H, 3.80 ppm, triplet, 4H, 3.91 ppm, quartet, 2H, 7.35 ppm, multiplet, 2H, 7.53 ppm, multiplet, 3H, 7.78 ppm, multiplet, 2H, 8.52 ppm, multiplet, 4H.

Example 73

[0862] ##STR00315##

[0863] To a stirred slurry of 2-phenyl-4,6-dichloro-5-phenylazopyrimidine (6.20 gm, 1.8810.sup.2 moles) in 2-butanol (75 mL) was added a solution of N-methyl-N-(2-aminoethyl)piperazine (6.0 gm, 4.1910.sup.2 moles) and diisopropylethylamine (5.41 gm, 4.1910.sup.2 moles) in 2-butanol (25 mL). This mixture was heated to boiling. The thick slurry which initially formed was transformed into a solution as the mixture reached reflux. The dark orange solution was heated at reflux for 5 hours. Upon cooling overnight at room temperature, the product separated as a yellow solid. The 2-butanol was removed under reduced pressure and the remaining solid was dissolved in methylene chloride (300 mL). This solution was washed with 10% potassium carbonate solution (150 mL) before being dried over magnesium sulfate. The solution was filtered and evaporated under reduced pressure. The remaining solid was stirred for 3 hours in diethyl ether (200 mL) and was then isolated by filtration, washed with diethyl ether and dried. The product was isolated in a yield of 5.6 gm, (55%).

##STR00316##

[0864] The phenylazopyrimidine (4.07 gm, 7.510.sup.2 moles) was stirred in methanol (40 mL) and THF (40 mL). To this was added 10% palladium on carbon (300 mg) and ammonium formate (3 gm) dissolved in water (6 mL). The solution was heated at 55 C. After being heated at 55 C. for 15 minutes, the orange color of the azopyrimidine had faded and TLC (silica, 25% methanol in methylene chloride) showed consumption of the azo compound with a single product. Water (10 mL) was added and stirring was continued for 5 minutes. After cooling, the catalyst was removed by filtration and the filtrates were treated with potassium carbonate until basic. The mixture was stirred with methylene chloride (200 mL). The methylene chloride phase was isolated and dried over magnesium sulfate. After filtration to remove the magnesium sulfate, the solvents were removed under reduced pressure. To the remaining material was added diethyl ether (50 mL) and hexane (50 mL) and this mixture was stirred for 5 minutes. The solvents were decanted from the crude triaminopyrimidine which was used without purification in the next step.

##STR00317##

[0865] The crude triaminopyrimidine (7.510.sup.3 moles) and ethyl benzoylformate (1.34 gm, 7.510.sup.3 moles) were combined in n-butanol (20 mL). This mixture was heated to 110 C. for 5 hours and then was kept at room temperature overnight. The n-butanol was removed under reduced pressure and the remaining material was purified by chromatography on silica using 15% methanol in methylene chloride as eluent. The fractions containing the product were pooled and evaporated under reduced pressure to yield 0.271 gm (6.4%) of the product. LC/MS: M+1=568.58.

Example 74

[0866] ##STR00318##

[0867] The triaminopyrimidine (0.855 gm, 2.010.sup.3 moles) and ethyl-3,5-difluorobenzoylformate (0.643 gm, 3.010.sup.3 moles) were combined in 2-butanol (10 mL). This mixture was heated at reflux for 5 hours. After cooling the pteridinone crystallized. The pteridinone was isolated by filtration, washed with 2-propanol followed by diethyl ether, and dried. The yield was 0.800 gm (69.2%). LC/MS: M+1=578.51.

[0868] TLR9 Antagonist Assay

[0869] HEK-Blue-hTLR9 cells were obtained from InvivoGen Inc. and used to determine test compound antagonism of human TLR9 (hTLR9) driven responses. HEK-Blue-hTLR9 cells are designed for studying the stimulation of human TLR9 by monitoring the activation of NF-kB. As described by the manufacturer, HEK-Blue-hTLR9 cells were obtained by co-transfection of the hTLR9 gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK293 cells. The SEAP reporter gene is placed under the control of the IFN-b minimal promoter fused to five NF-kB and AP-1 binding sites. Stimulation with a TLR9 ligand activates NF-kB and AP-1 which induces the production of SEAP. Levels of SEAP can be easily determined with QUANTI-Blue a detection medium that turns purple/blue in the presence of alkaline phosphatase.

[0870] TLR9 Antagonism Assay

[0871] Day 1:

[0872] A cell suspension of HEK-Blue-hTLR9 cells at 450,000 cells per ml in test medium which contained 5% (v/v) heat inactivated FBS was prepared. 180 ul of cell suspension (80,000 cells) was added per well of a flat-bottom 96-well plate and place in an incubator at 37 C. for overnight.

[0873] Day 2

[0874] Test compounds were serially diluted in test medium, generally starting at 10 uM, and diluting by 3 fold in a 96 well master plate. 20 ul of diluted test compound was transferred using a 12 channel multi-channel pipet to the cell plate and incubated at 37 C. for 1 hour. Then 20 ul of an hTLR9 agonist (such as ODN 2006, 1 uM) was added to each well and the plate incubated at 37 C. overnight.

[0875] Day 3

[0876] Invivogen's QUANTI-Blue was prepared following the manufacturer's instructions. 180 ml of resuspended QUANTI-Blue was added per well of a flat bottom 96-well plate. 20 ul per well of induced HEK-Blue-hTLR9 cells supernatant was then added to the plate and the plate was incubated at 37 C. for 1-3 h. SEAP levels were determined using a spectrophotometer at 620 nm.

[0877] Calculation of IC.sub.50

[0878] The concentration dependent inhibition of hTLR9 dependent SEAP production was expressed as the concentration of compound which produced half the maximal level of SEAP induced by the hTLR agonist alone. Percent activity was calculated for each observation using the formula: % activity=((observed O.D.background O.D.)/(agonist only O.D.background O.D.))*100. The 50% inhibitory concentration (IC.sub.50) was calculated by using a 4 parameter Hill plot sigmoidal curve fit where the inflection point of the sigmoidal curve is defined as the point of 50% activity. The results are shown in Table 4.

TABLE-US-00004 TABLE 4 hTLR9 antagonism Examples IC.sub.50 (nM) Example 1 285 Example 56 317 Example 54 62 Example 69 2480 Example 70 137 Example 71 3821 Example 72 487 Example 73 124 Example 74 901 Example 57 113 Example 59 991 Example 60 2184 Example 61 >10000 Example 62 2493 Example 63 112 Example 64 2950 Example 65 198 Example 67 25 Example 66 181 Example 68 273

[0879] The Effects of Test Articles on Toll-Like Receptor (TLR) Knockdown Following a Single Intraperitoneal Dose to Male C57Bl/6 Mice.

[0880] Toll-Like Receptor (TLR) knockdown effect of test articles was evaluated in a C57Bl/6J mouse. Primary end points included a terminal blood collection for analysis of cytokine production in response to CpG-DNA TLR9 agonist injection. Male C57Bl/6J mice, at 8 weeks of age from Jackson Laboratories were used. Test groups were 3 mice per treatment group and the groups were administered test article in a series of descending doses within the range of 400 g to 10 g. The results are shown in Table 5. Test article treatment was dosed at T=0 hr by intraperitoneal injection. Agonist (CpG ODN 1668) treatment was dosed one hour later, T=1 hr by intraperitoneal injection. Necropsy was performed 3 hours post agonist treatment, T=4 hr. Blood samples were collected into serum separator tubes, allowed to clot at room temperature for at least 20 minutes, centrifuged at ambient temperature at 3000 g for 10 minutes, and the serum was extracted. ELISA was performed to determine murine IL-12 levels following manufacture's protocol (BioLegend Inc.). Serum IL-12 levels were calculated and plotted versus administered dose of antagonist and inhibitory dose at 50% (ID.sub.50) was determined.

TABLE-US-00005 TABLE 5 In vivo TLR antagonism Examples g ID.sub.50 Example 1 76 Example 56 86 Example 54 33 Example 73 128 Example 57 36