BICYCLIC HETEROARENES AND METHODS OF THEIR USE

Abstract

Disclosed are compounds useful in the treatment of neurological disorders. The compounds described herein, alone or in combination with other pharmaceutically active agents, can be used for treating or preventing neurological diseases.

Claims

1. A compound of formula (I): ##STR00279## or a pharmaceutically acceptable salt thereof, wherein is a single bond, X.sup.1 is (C(R.sup.A).sub.2).sub.m or —OC(R.sup.A).sub.2—R.sup.X, and X.sup.2 is C(R.sup.A).sub.2 or CO; is a double bond, and each of X.sup.1 and X.sup.2 is independently CR.sup.A or N, wherein R.sup.X is a bond to X.sup.2; R.sup.1 is -(L).sub.n-R.sup.1; optionally substituted C.sub.1-9 alkoxy; optionally substituted C.sub.1-9 heterocyclyl comprising at least one endocyclic oxygen; unsubstituted pyrimidinyl; optionally substituted pyridazinyl; optionally substituted oxazolyl, or pyrid-2-on-1-yl; R.sup.2 is optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heterocyclyl, or optionally substituted C.sub.1-9 heteroaryl; R.sup.3 is a group of the following structure: ##STR00280## each R.sup.A is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; R.sup.B is optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heterocyclyl; R.sup.C is H or optionally substituted C.sub.1-6 alkyl; each L is independently optionally substituted C.sub.1-6 alkylene, O, or NR.sup.C; n is 1, 2, or 3; and m is 0, 1, or 2.

2. The compound of claim 1, wherein is a single bond.

3. The compound of claim 1 or 2, wherein X.sup.1 is (C(R.sup.A).sub.2).sub.m.

4. The compound of claim 3, wherein m is 1.

5. The compound of any one of claims 1 to 4, wherein X.sup.2 is C(R.sup.A).sub.2.

6. The compound of any one of claims 1 to 5, wherein each R.sup.A is hydrogen.

7. The compound of claim 1, wherein the compound is of formula (Ia): ##STR00281## or a pharmaceutically acceptable salt thereof.

8. The compound of claim 1, wherein the compound is of formula (Ia′): ##STR00282## or a pharmaceutically acceptable salt thereof.

9. The compound of claim 1, wherein the compound is of formula (Ib): ##STR00283## or a pharmaceutically acceptable salt thereof.

10. The compound of claim 1, wherein the compound is of formula (Ic): ##STR00284## or a pharmaceutically acceptable salt thereof.

11. The compound of claim 1, wherein the compound is of formula (Id): ##STR00285## or a pharmaceutically acceptable salt thereof.

12. The compound of claim 1, wherein the compound is of formula (Ie): ##STR00286## or a pharmaceutically acceptable salt thereof.

13. The compound of any one of claims 1 to 12, wherein R.sup.1 is —O-(L).sub.(n-1)-R.sup.B.

14. The compound of any one of claims 1 to 13, wherein n is 2.

15. The compound of any one of claims 1 to 13, wherein n is 1.

16. The compound of any one of claims 1 to 15, wherein at least one L is optionally substituted C.sub.1-6 alkylene.

17. The compound of claim 16, wherein the optionally substituted C.sub.1-6 alkylene is methylene.

18. The compound of claim 16, wherein the optionally substituted C.sub.1-6 alkylene is ethylene.

19. The compound of any one of claims 1 to 18, wherein R.sup.B is optionally substituted non-aromatic C.sub.1-9 heterocyclyl.

20. The compound of any one of claims 1 to 18, wherein R.sup.B is optionally substituted C.sub.1-9 heteroaryl.

21. The compound of any one of claims 1 to 18, wherein R.sup.B is optionally substituted C.sub.1-6 alkyl.

22. The compound of any one of claims 1 to 12, wherein R.sup.1 is: ##STR00287## ##STR00288## or methoxy.

23. The compound of claim 22, wherein R.sup.1 is: ##STR00289##

24. The compound of any one of claims 1 to 23, wherein R.sup.2 is: ##STR00290##

25. The compound of claim 24, wherein R.sup.2 is: ##STR00291##

26. The compound of any one of claims 1 to 25, wherein R.sup.3 is: ##STR00292##

27. A compound of the following structure: ##STR00293## ##STR00294## ##STR00295## ##STR00296## ##STR00297## ##STR00298## ##STR00299## ##STR00300## or a pharmaceutically acceptable salt thereof.

28. A compound of the following structure: ##STR00301## ##STR00302## ##STR00303## ##STR00304## ##STR00305## ##STR00306## ##STR00307## ##STR00308## ##STR00309## ##STR00310## ##STR00311## ##STR00312## ##STR00313## ##STR00314## ##STR00315## ##STR00316## ##STR00317## ##STR00318## ##STR00319## or a pharmaceutically acceptable salt thereof.

29. A pharmaceutical composition comprising the compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

30. A method of treating a neurological disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the compound of any one of claims 1 to 28, or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 29.

31. The method of claim 30, wherein the neurological disorder is FTLD-TDP, chronic traumatic encephalopathy, ALS, Alzheimer's disease, LATE, or frontotemporal lobar degeneration.

32. The method of claim 31, wherein the neurological disorder is ALS.

33. A method of inhibiting toxicity in a cell related to a protein, the method comprising contacting the cell with the compound of any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof.

34. The method of claim 33, wherein the toxicity is TDP-43-related toxicity.

35. The method of claim 33, wherein the toxicity is C9orf72-related toxicity.

36. A method of inhibiting PIKfyve in a cell expressing PIKfyve protein, the method comprising contacting the cell with the compound of any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof.

37. The method of any one of claims 33 to 36, wherein the cell is a mammalian neural cell.

38. The method of any one of claims 33 to 37, wherein the cell is in a subject.

39. The method of claim 38, wherein the subject suffers from a neurological disorder.

40. A method of treating a TDP-43-associated disorder in a subject, the method comprising administering to the subject in need thereof an effective amount of the compound of formula (I): ##STR00320## or a pharmaceutically acceptable salt thereof, wherein is a single bond, X.sup.1 is (C(R.sup.A).sub.2).sub.m or —OC(R.sup.A).sub.2—R.sup.X, and X.sup.2 is C(R.sup.A).sub.2 or CO; or is a double bond, and each of X.sup.1 and X.sup.2 is independently CR.sup.A or N, wherein R.sup.X is a bond to X.sup.2; R.sup.1 is -(L).sub.n-R.sup.B; hydrogen; halogen; cyano; optionally substituted C.sub.1-6 alkyl; optionally substituted C.sub.1-6 heteroalkyl; optionally substituted C.sub.1-6 alkoxy; optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heterocyclyl, or optionally substituted C.sub.1-9 heteroaryl; R.sup.2 is hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heterocyclyl, or optionally substituted C.sub.1-9 heteroaryl; R.sup.3 is a group of the following structure: ##STR00321## each R.sup.A is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; R.sup.B is optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heterocyclyl; R.sup.C is H or optionally substituted C.sub.1-6 alkyl; each L is independently optionally substituted alkylene, O, or NR.sup.C; and n is 1, 2, or 3; and m is 0, 1, or 2.

41. The method of claim 40, wherein is a single bond.

42. The method of claim 40 or 41, wherein X.sup.1 is (C(R.sup.A).sub.2).sub.m.

43. The method of claim 42, wherein m is 1.

44. The method of any one of claims 40 to 43, wherein X.sup.2 is C(R.sup.A).sub.2.

45. The method of any one of claims 40 to 44, wherein each R.sup.A is hydrogen.

46. The method of claim 40, wherein the compound is of formula (Ia): ##STR00322## or a pharmaceutically acceptable salt thereof.

47. The method of claim 40, wherein the compound is of formula (Ia′): ##STR00323## or a pharmaceutically acceptable salt thereof.

48. The method of claim 40, wherein the compound is of formula (Ib): ##STR00324## or a pharmaceutically acceptable salt thereof.

49. The method of claim 40, wherein the compound is of formula (Ic): ##STR00325## or a pharmaceutically acceptable salt thereof.

50. The method of claim 40, wherein the compound is of formula (Id): ##STR00326## or a pharmaceutically acceptable salt thereof.

51. The method of claim 40, wherein the compound is of formula (Ie): ##STR00327## or a pharmaceutically acceptable salt thereof.

52. The method of any one of claims 40 to 51, wherein R.sup.1 is —O-(L).sub.(n-1)-R.sup.B.

53. The method of any one of claims 40 to 52, wherein n is 2.

54. The method of any one of claims 40 to 52, wherein n is 1.

55. The method of any one of claims 40 to 53, wherein at least one L is optionally substituted C.sub.1-6 alkylene.

56. The method of claim 55, wherein the optionally substituted C.sub.1-6 alkylene is methylene.

57. The method of claim 55, wherein the optionally substituted C.sub.1-6 alkylene is ethylene.

58. The method of any one of claims 40 to 57, wherein R.sup.B is optionally substituted non-aromatic C.sub.1-9 heterocyclyl.

59. The method of any one of claims 40 to 57, wherein R.sup.B is optionally substituted C.sub.1-9 heteroaryl.

60. The method of any one of claims 40 to 57, wherein R.sup.B is optionally substituted C.sub.1-6 alkyl.

61. The method of any one of claims 40 to 60, wherein R.sup.1 is: ##STR00328## ##STR00329## ##STR00330## ##STR00331## hydrogen, chloro, methyl, cyano, or methoxy.

62. The method of claim 61, wherein R.sup.1 is: ##STR00332## or methoxy.

63. The method of any one of claims 40 to 62, wherein R.sup.2 is: ##STR00333##

64. The method of claim 51, wherein R.sup.2 is: ##STR00334##

65. The method of any one of claims 40 to 64, wherein R.sup.3 is: ##STR00335##

66. The method of claim 65, wherein the compound is a compound of claim 27 or 28 or a pharmaceutically acceptable salt thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0162] FIG. 1 is a scheme showing an approach to generation of a control TDP-43 yeast model (FAB1 TDP-43). A control yeast TDP-43 model was generated by integrating the human TDP-43 gene and the GAL1 promoter into the yeast genome. The yeast ortholog of human PIKFYVE is FAB1.

[0163] FIG. 2 is a scheme showing an approach to generation of a humanized PIKFYVE TDP-43 yeast model (PIKFYVE TDP-43). FAB1 gene through homologous recombination with a G418 resistance cassette (fab1::G418.sup.R) (FIG. 2). PIKFYVE was cloned downstream of the GPD promoter harbored on a URA3-containing plasmid and introduced into the fab1::G418R ura3 strain. The pGAL1-TDP-43 construct was then introduced into the “humanized” yeast strain and assessed for cytotoxicity.

[0164] FIG. 3 is a histogram generated from the flow cytometry-based viability assay of FAB1 TDP-43.

[0165] FIG. 4 is a histogram generated from the flow cytometry-based viability assay of PIKFYVE TDP-43. Upon induction of TDP-43, there was a marked increase in inviable cells (rightmost population), with a more pronounced effect in PIKFYVE TDP-43 than in FAB1 TDP-43 strain (see FIG. 3).

[0166] FIG. 5 is an overlay of histograms generated from the flow cytometry-based viability assay of FAB1 TDP-43 in the presence of APY0201.

[0167] FIG. 6 is an overlay of histograms generated from the flow cytometry-based viability assay of PIKFYVE TDP-43 in the presence of APY0201.

[0168] FIG. 7 is a scatter plot comparing cytoprotection efficacy in PIKFYVE TDP-43 to PIKfyve inhibitory activity of test compounds.

DETAILED DESCRIPTION

[0169] The present invention features compositions and methods for treating neurological disorders, such as amyotrophic lateral sclerosis and other neuromuscular disorders, as well as frontotemporal degeneration, Alzheimer's disease, Parkinson's disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, Inclusion body myopathy with early-onset Paget disease and frontotemporal dementia (IBMPFD), sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy among others. Particularly, the invention provides inhibitors of FYVE-type zinc finger containing phosphoinositide kinase (PIKfyve), that may be administered to a patient (e.g., a human patient) so as to treat or prevent a neurological disorder, such as one or more of the foregoing conditions. In the context of therapeutic treatment, the PIKfyve inhibitor may be administered to the patient to alleviate one or more symptoms of the disorder and/or to remedy an underlying molecular pathology associated with the disease, such as to suppress or prevent aggregation of TAR-DNA binding protein (TDP)-43.

[0170] The disclosure herein is based, in part, on the discovery that PIKfyve inhibition modulates TDP-43 aggregation in cells. Suppression of TDP-43 aggregation exerts beneficial effects in patients suffering from a neurological disorder. Many pathological conditions have been correlated with TDP-43-promoted aggregation and toxicity, such as amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer's disease, Parkinson's disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy. Without being limited by mechanism, by administering an inhibitor of PIKfyve, patients suffering from diseases associated with TDP-43 aggregation and toxicity may be treated, for example, due to the suppression of TDP-43 aggregation induced by the PIKfyve inhibitor.

[0171] Patients that are likely to respond to PIKfyve inhibition as described herein include those that have or are at risk of developing TDP-43 aggregation, such as those that express a mutant form of TDP-43 associated with TDP-43 aggregation and toxicity in vivo. Examples of such mutations in TDP-43 that have been correlated with elevated TDP-43 aggregation and toxicity include Q331K, M337V, Q343R, N345K, R361S, and N390D, among others. The compositions and methods described herein thus provide the additional clinical benefit of enabling the identification of patients that are likely to respond to PIKfyve inhibitor therapy, as well as processes for treating these patients accordingly.

[0172] The sections that follow provide a description of exemplary PIKfyve inhibitors that may be used in conjunction with the compositions and methods disclosed herein. The sections below additionally provide a description of various exemplary routes of administration and pharmaceutical compositions that may be used for delivery of these substances for the treatment of a neurological disorder.

PIKfyve Inhibitors

[0173] Exemplary PIKfyve inhibitors described herein include compounds of formula (I)

##STR00148## [0174] or a pharmaceutically acceptable salt thereof, [0175] where [0176] is a single bond, X.sup.1 is C(R.sup.A).sub.2 or —OC(R.sup.A).sub.2—R.sup.X, and X.sup.2 is C(R.sup.A).sub.2 or CO; or is a double bond, and each of X.sup.1 and X.sup.2 is independently CR.sup.A or N, wherein R.sup.X is a bond to X.sup.2; [0177] R.sup.1 is -(L).sub.n-R.sup.B; hydrogen; halogen; cyano; optionally substituted C.sub.1-6 alkyl; optionally substituted C.sub.1-6 heteroalkyl; optionally substituted C.sub.1-6 alkoxy; optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heterocyclyl, or optionally substituted C.sub.1-9 heteroaryl; [0178] R.sup.2 is hydrogen, optionally substituted C.sub.1-6 alkyl, optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heterocyclyl, or optionally substituted C.sub.1-9 heteroaryl; [0179] R.sup.3 is a group of the following structure:

##STR00149## [0180] each R.sup.A is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; [0181] R.sup.B is optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heteroaryl, optionally substituted C.sub.3-8 cycloalkyl, or optionally substituted C.sub.1-9 heterocyclyl; [0182] R.sup.C is H or optionally substituted C.sub.1-6 alkyl; [0183] each L is independently optionally substituted alkylene, O, or NR.sup.C; and [0184] n is 1, 2, or 3.

[0185] In some embodiments, R.sup.1 is -(L).sub.n-R.sup.B; optionally substituted C.sub.1-6 alkoxy; optionally substituted C.sub.1-9 heterocyclyl comprising at least one endocyclic oxygen; unsubstituted pyrimidinyl; optionally substituted pyridazinyl; optionally substituted oxazolyl, or pyrid-2-on-1-yl. In some embodiments, R.sup.2 is optionally substituted C.sub.6-10 aryl, optionally substituted C.sub.1-9 heterocyclyl, or optionally substituted C.sub.1-9 heteroaryl.

[0186] Methods of Treatment

[0187] Suppression of PIKfyve Activity and TDP-43 Aggregation to Treat Neurological Disorders

[0188] Using the compositions and methods described herein, a patient suffering from a neurological disorder may be administered a PIKfyve inhibitor, such as a small molecule described herein, so as to treat the disorder and/or to suppress one or more symptoms associated with the disorder. Exemplary neurological disorders that may be treated using the compositions and methods described herein are, without limitation, amyotrophic lateral sclerosis, frontotemporal degeneration, Alzheimer's disease, Parkinson's disease, dementia with Lewy Bodies, corticobasal degeneration, progressive supranuclear palsy, dementia parkinsonism ALS complex of Guam, Huntington's disease, IBMPFD, sporadic inclusion body myositis, myofibrillar myopathy, dementia pugilistica, chronic traumatic encephalopathy, Alexander disease, and hereditary inclusion body myopathy, as well as neuromuscular diseases such as congenital myasthenic syndrome, congenital myopathy, cramp fasciculation syndrome, Duchenne muscular dystrophy, glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis, Isaac's Syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal and bulbar muscular atrophy, spinal muscular atrophy, Stiff person syndrome, Troyer syndrome, and Guillain-Barré syndrome.

[0189] The present disclosure is based, in part, on the discovery that PIKfyve inhibitors, such as the agents described herein, are capable of attenuating TDP-43 toxicity. TDP-43-promoted toxicity has been associated with various neurological diseases. The discovery that PIKfyve inhibitors modulate TDP-43 aggregation provides an important therapeutic benefit. Using a PIKfyve inhibitor, such as a PIKfyve inhibitor described herein, a patient suffering from a neurological disorder or at risk of developing such a condition may be treated in a manner that remedies an underlying molecular etiology of the disease. Without being limited by mechanism, the compositions and methods described herein can be used to treat or prevent such neurological conditions, for example, by suppressing the TDP-43 aggregation that promotes pathology.

[0190] Additionally, the compositions and methods described herein provide the beneficial feature of enabling the identification and treatment of patients that are likely to respond to PIKfyve inhibitor therapy. For example, in some embodiments, a patient (e.g., a human patient suffering from or at risk of developing a neurological disease described herein, such as amyotrophic lateral sclerosis) is administered a PIKfyve inhibitor if the patient is identified as likely to respond to this form of treatment. Patients may be identified as such on the basis, for example, of susceptibility to TDP-43 aggregation. In some embodiments, the patient is identified is likely to respond to PIKfyve inhibitor treatment based on the isoform of TDP-43 expressed by the patient. For example, patients expressing TDP-43 isoforms having a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D, among others, are more likely to develop TDP-43-promoted aggregation and toxicity relative to patients that do not express such isoforms of TDP-43. Using the compositions and methods described herein, a patient may be identified as likely to respond to PIKfyve inhibitor therapy on the basis of expressing such an isoform of TDP-43, and may subsequently be administered a PIKfyve inhibitor so as to treat or prevent one or more neurological disorders, such as one or more of the neurological disorders described herein.

[0191] Assessing Patient Response

[0192] A variety of methods known in the art and described herein can be used to determine whether a patient having a neurological disorder (e.g., a patient at risk of developing TDP-43 aggregation, such as a patient expressing a mutant form of TDP-43 having a mutation associated with elevated TDP-43 aggregation and toxicity, for example, a mutation selected from Q331K, M337V, Q343R, N345K, R361S, and N390D) is responding favorably to PIKfyve inhibition. For example, successful treatment of a patient having a neurological disease, such as amyotrophic lateral sclerosis, with a PIKfyve inhibitor described herein may be signaled by: [0193] (i) an improvement in condition as assessed using the amyotrophic lateral sclerosis functional rating scale (ALSFRS) or the revised ALSFRS (ALSFRS-R), such as an improvement in the patient's ALSFRS or ALSFRS-R score within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., an improvement in the patient's ALSFRS or ALSFRS-R score within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient); (ii) an increase in slow vital capacity, such as an increase in the patient's slow vital capacity within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., an increase in the patient's slow vital capacity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient); [0194] (iii) a reduction in decremental responses exhibited by the patient upon repetitive nerve stimulation, such as a reduction that is observed within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., a reduction that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient); [0195] (iv) an improvement in muscle strength, as assessed, for example, by way of the Medical Research Council muscle testing scale (as described, e.g., in Jagtap et al., Ann. Indian. Acad. Neurol. 17:336-339 (2014), the disclosure of which is incorporated herein by reference as it pertains to measuring patient response to neurological disease treatment), such as an improvement that is observed within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., an improvement that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient); [0196] (v) an improvement in quality of life, as assessed, for example, using the amyotrophic lateral sclerosis-specific quality of life (ALS-specific QOL) questionnaire, such as an improvement in the patient's quality of life that is observed within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., an improvement in the subject's quality of life that is observed within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient); [0197] (vi) a decrease in the frequency and/or severity of muscle cramps, such as a decrease in cramp frequency and/or severity within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., a decrease in cramp frequency and/or severity within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient); and/or [0198] (vii) a decrease in TDP-43 aggregation, such as a decrease in TDP-43 aggregation within one or more days, weeks, or months following administration of the PIKfyve inhibitor (e.g., a decrease in TDP-43 aggregation within from about 1 day to about 48 weeks (e.g., within from about 2 days to about 36 weeks, from about 4 weeks to about 24 weeks, from about 8 weeks to about 20 weeks, or from about 12 weeks to about 16 weeks), or more, following the initial administration of the PIKfyve inhibitor to the patient, such as within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, or more, following the initial administration of the PIKfyve inhibitor to the patient.

Combination Formulations and Uses Thereof

[0199] The compounds of the invention can be combined with one or more therapeutic agents. In particular, the therapeutic agent can be one that treats or prophylactically treats any neurological disorder described herein.

[0200] Combination Therapies

[0201] A compound of the invention can be used alone or in combination with other agents that treat neurological disorders or symptoms associated therewith, or in combination with other types of treatment to treat, prevent, and/or reduce the risk of any neurological disorders. In combination treatments, the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6, 2005). In this case, dosages of the compounds when combined should provide a therapeutic effect.

Pharmaceutical Compositions

[0202] The compounds of the invention are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. Accordingly, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention in admixture with a suitable diluent, carrier, or excipient.

[0203] The compounds of the invention may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the scope of the invention. In accordance with the methods of the invention, the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0204] A compound of the invention may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a compound of the invention may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers.

[0205] A compound of the invention may also be administered parenterally. Solutions of a compound of the invention can be prepared in water suitably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003, 20.sup.th ed.) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19), published in 1999.

[0206] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe.

[0207] Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter.

[0208] The compounds of the invention may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.

Dosages

[0209] The dosage of the compounds of the invention, and/or compositions comprising a compound of the invention, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds of the invention are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form). Dose ranges include, for example, between 10-1000 mg.

[0210] Alternatively, the dosage amount can be calculated using the body weight of the patient. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1-50 mg/kg.

[0211] The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way.

EXAMPLES

List of Abbreviations:

[0212] DIPEA=N,N-diisopropylethylamine [0213] EtOH=ethanol [0214] THF=tetrahydrofuran [0215] nBuLi=n-butyl lithium [0216] I.sub.2=iodine [0217] Pd(dppf)Cl.sub.2=[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) [0218] Cs.sub.2CO.sub.3=cesium carbonate [0219] H.sub.2O=water [0220] Pd(PPh.sub.3)Cl.sub.2=Bis(triphenylphosphine)palladium(II) dichloride [0221] Pd(PPh.sub.3).sub.4=tetrakis(triphenylphosphine)palladium(0) [0222] LiCl=lithium chloride [0223] MecOH=methanol [0224] NBS=N-bromosuccinimide [0225] ACN=acetonitrile [0226] K.sub.2CO.sub.3=potassium carbonate [0227] DMA=N,N-dimethylacetamide [0228] Zn(CN).sub.2=zinc cyanide [0229] HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0230] DMF=N,N-dimethylformamide [0231] Pd(t-Bu.sub.3P).sub.2=Bis(tri-tert-butylphosphine)palladium(0) [0232] DMF-DMA=N,N-dimethylformamide dimethyl acetal [0233] N.sub.2H.sub.4H.sub.2O=hydrazine hydrate [0234] Pd.sub.2(dba).sub.3=tris(dibenzylideneacetone) dipalladium [0235] X-Phos=2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl [0236] Pd(PPh.sub.3)Cl.sub.2 DCM=Bis(triphenylphosphine)palladium(II) dichloride dichloromethane complex [0237] DMSO=dimethylsulfoxide [0238] DPPA=diphenyl phosphorylazide [0239] Et.sub.3N=triethylamine [0240] HCl=hydrochloric acid

Example 1. Preparation of Compounds

General Schemes

[0241] ##STR00150##

[0242] An appropriately substituted morpholine I can be reacted with an appropriated substituted aryl alcohol II under basic conditions to afford an appropriately substituted morpholine ether intermediate III. The morpholine ether intermediate III is then coupled with an appropriately substituted aryl amine IV under Buchwald-Hartwig conditions to yield an appropriately substituted aryl morpholine ether product V.

##STR00151##

[0243] An appropriately substituted dihydrofuran VI is reacted with an appropriately substituted carbonate VII under basic conditions to afford the appropriately substituted tetrahydrofuran ester intermediate VIII. An appropriately substituted morpholine imine intermediate IX is either (A) reacted with the tetrahydrofuran ester intermediate VIII under Claisen condensation conditions or (B) reacted with phosphoryl chloride X under basic conditions to afford the appropriately substituted pyrimidine morpholine intermediate XI. This pyrimidine morpholine intermediate XI is reacted with an appropriately substituted aryl amine XII under basic conditions to afford the appropriately substituted pyrrolopyrimidine product XIII.

Synthesis of 4-(7-phenyl-4-(2-(pyridin-2-yl)ethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 1)

[0244] ##STR00152##

Step 1: Synthesis of 4-(4-chloro-5-(2-chloroethyl)-6-(2-(pyridin-2-yl)ethoxy)pyrimidin-2-yl)morpholine

[0245] A solution of 2-(pyridin-2-yl)ethanol (830 mg, 6.74 mmol) in DMF was added to a solution of sodium hydride (270 mg, 6.74 mmol) in DMF (60 mL) at 0° C. The resultant mixture was warmed up and stirred at room temperature for 10 min followed by the addition of 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (2 g, 6.74 mmol). The reaction mixture was stirred further at room temperature for 48 h. It was quenched with water (200 mL) and extracted with ethyl acetate (300 mL×2). The combined organic layers were washed with water (200 mL×2), brine (200 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography, eluting with petroleum ether/ethyl acetate=3/1 to obtain 4-(4-chloro-5-(2-chloroethyl)-6-(2-(pyridin-2-yl)ethoxy)pyrimidin-2-yl)morpholine (1.9 g, 74%) as off-white solid. LCMS (ESI) m/z: 398.1 [M+16].sup.+.

Step 2: Synthesis of 4-(7-phenyl-4-(2-(pyridin-2-yl)ethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0246] A mixture of 4-(4-chloro-5-(2-chloroethyl)-6-(2-(pyridin-2-yl)ethoxy)pyrimidin-2-yl)morpholine (100 mg, 0.261 mmol), aniline (49 mg, 0.526 mmol), tris(dibenzylideneacetone)dipalladium (24 mg, 0.026 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (30 mg, 0.052 mmol) and cesium carbonate (170 mg, 0.522 mmol) in dioxane (5 mL) was stirred at 100° C. for 16 h under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (100 mL), washed with water (30 mL×2), brine (30 mL), dried over sodium sulfate, filtered and concentrated. The residue obtained was subjected to silica gel column chromatography (eluting with petroleum ether/ethyl acetate=2/1) and then to PREP-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 4-(7-phenyl-4-(2-(pyridin-2-yl)ethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (53.8 mg, 51%) as white solid. .sup.1H NMR (400 MHz, DMSO-de) b 8.50 (dd, J=4.8, 0.8 Hz, 1H), 7.75-7.70 (m, 2H), 7.35-7.31 (m, 3H), 7.25-7.22 (m, 1H), 6.96 (t, J=7.2 Hz, 1H), 4.63 (t, J=6.8 Hz, 2H), 3.99 (t, J=8.6 Hz, 2H), 3.66 (s, 8H), 3.16 (t, J=6.8 Hz, 2H), 2.79 (t, J=8.6 Hz, 2H). LCMS (ESI) m/z: 404.2 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-(pyridin-2-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 2)

[0247] ##STR00153##

[0248] To a solution of pyridin-2-ylmethanol (52 mg, 0.47 mmol) in dry THE (10 mL) was added NaH (28 mg, 0.71 mmol) and the mixture was stirred at 0° C. 15 min. A solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (150 mg, 0.47 mmol) in THE (5 mL) was then added and the resultant mixture stirred for another 16 h at 100° C. The reaction was quenched with ice water (20 mL) and extracted with EtOAc (20 mL*3). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by prep-HPLC to give 4-(7-phenyl-4-(pyridin-2-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (28.4 mg, 16%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.54 (dd, J=4.8, 0.8 Hz, 1H), 7.82-7.78 (m, 1H), 7.76 (s, 1H), 7.74 (s, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.36-7.29 (m, 3H), 6.97 (t, J=7.2 Hz, 1H), 5.44 (s, 2H), 4.04 (t, J=8.8 Hz, 2H), 3.59 (s, 8H), 2.94 (t, J=8.8 Hz, 2H); LCMS (ESI) m/z:390.3 [M+H].sup.+.

[0249] The following compounds were synthesized according to the protocol described above:

TABLE-US-00003 Compound # Structure LCMS NMR  3 [00154] LCMS (ESI) m/z: 390 [M + H].sup.+. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.53 (s, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.30-7.50 (m, 3H), 6.98 (m, 1H), 5.44 (s, 2H), 4.03 (t, J = 8.0 Hz, 2H), 3.66 (s, 8H), 2.90 (t, J = 8.4 Hz, 2H)  4 [00155] LCMS (ESI) m/z: 390 [M + H].sup.+ 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 2H), 7.75 (d, J = 6.4 Hz, 2H), 7.25-7.45 (m, 4H), 6.98 (t, J = 6.0 Hz, 1H), 5.44 (s, 2H), 4.06 (t, J = 2.8 Hz, 2H), 3.61 (s, 8H), 2.96 (t, J = 3.2 Hz, 2H)  5 [00156] LCMS (ESI) m/z: 391.2 [M + H].sup.+. 1H NMR (500 MHz, DMSO-d6) δ 2.95 (t, J = 9.0 Hz, 2H), 3.57 (s, 8H), 4.05 (t, J = 8.0 Hz, 2H), 5.65 (s, 2H), 6.97 (t, J = 7.5 Hz, 1H), 7.34 (q, J = 7.0 Hz, 2H), 7.70 (d, J = 3.5 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 9.17 (t, J = 3.0 Hz, 1H)  6 [00157] LCMS (ESI) m/z: 391 [M + H].sup.+. 1H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.67 (s, 1H), 8.54 (s, 1H), 8.18 (m, 2H), 7.86 (m, 1H), 7.40 (m, 2H), 5.45 (s, 2H), 4.06 (t, J = 8.4 Hz, 2H) 3.67 (s, 8H), 2.93 (t, J = 8.4 Hz, 2H)  7 [00158] LCMS (ESI) m/z: 410.3 [M + H].sup.+ 1H NMR (500 MHz, DMSO-d6) δ 8.22 (s, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 6.97 (t, J = 7.5 Hz, 1H), 4.22-4.19 (m, 1H), 4.15-4.12 (m, 1H), 4.02 (t, J = 8.5 Hz, 2H), 3.66 (s, 8H), 2.88 (t, J = 9.0 Hz, 2H), 2.83 (d, J = 10.5 Hz, 1H), 2.70 (d, J = 10.5 Hz, 1H), 2.21 (s, 3H), 1.98 (t, J = 10.0 Hz, 2H), 1.85 (t, J = 10.0 Hz, 1H), 1.70-1.63 (m, 2H), 1.53-1.49 (m, 1H), 1.08-1.01 (m, 1H)  8 [00159] LCMS (ESI) m/z: 383.1 [M + H].sup.+ 1H NMR (400 MHz, DMSO-d6) δ 7.76-7.74 (d, J = 8.0 Hz, 2H), 7.36-7.32 (m, 2H), 6.97 (m, 1H), 4.28-4.21 (m, 2H), 4.13 (m, 1H), 4.01 (t, J = 8.6 Hz, 2H), 3.80-3.75 (m, 1H), 3.69-3.64 (m, 9H), 2.86 (t, J = 8.6 Hz, 2H), 1.97-1.81 (m, 3H), 1.63 (m, 1H)  9 [00160] LCMS (ESI) m/z: 397.2 [M + H].sup.+ 1H NMR (500 MHz, DMSO-d6) δ 7.75 (d, J = 5.0 Hz, 2H), 7.34 (t, J = 8.0 Hz, 2H), 6.96 (t, J = 7.5 Hz, 1H), 4.42-4.29 (m, 2H), 4.01 (t, J = 8.5 Hz, 2H), 3.90-3.84 (m, 1H), 3.75 (dd, J = 14.0, 7.5 Hz, 1H), 3.66 (s, 8H), 3.62-3.58 (m, 1H), 2.86 (t, J = 8.0 Hz, 2H), 2.00-1.97 (m, 1H), 1.88-1.80 (m, 4 H), 1.49-1.45 (m, 1H) 10 [00161] LCMS (ESI) m/z: 398.1 [M + H].sup.+. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (d, J = 2.4 Hz, 1H), 8.18-8.14 (m, 2H), 7.36 (m, 1H), 4.41-4.31 (m, 2H), 4.04 (t, J = 8.6 Hz, 2H), 3.87 (m, 1H), 3.78-3.57 (m, 10H), 2.89 (t, J = 8.6 Hz, 2H), 1.98 (m, 1H), 1.88-1.79 (m, 2H), 1.47 (m, 1H)

Synthesis of tert-butyl 4-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate (Compound 28) and 4-(7-(piperidin-4-yl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 53)

[0250] ##STR00162##

Step 1: Synthesis of tert-butyl 4-(6-chloro-5-(2-chloroethyl)-2-morpholinopyrimidin-4-ylamino)piperidine-1-carboxylate

[0251] To a stirred solution of 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (100 mg, 0.337 mmol) and tert-butyl 4-aminopiperidine-1-carboxylate (135 mg, 0.674 mmol) in acetonitrile (5 mL) at room temperature was added DIPEA (109 mg, 0.843 mmol) and the resultant mixture was refluxed for 16 h. After cooling to room temperature, the mixture was diluted with ethyl acetate (50 mL), washed with water (20 mL), brine (20 mL), dried over sodium sulfate, filtered and concentrated to obtain tert-butyl 4-(6-chloro-5-(2-chloroethyl)-2-morpholinopyrimidin-4-ylamino)piperidine-1-carboxylate (100 mg, 64%) as white solid. This material was used in the next step without further purification. LCMS (ESI) m/z: 460.1 [M+H].sup.+.

Step 2: Synthesis of tert-butyl 4-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate

[0252] Cesium carbonate (177 mg, 0.543 mmol) was added to a mixture of tert-butyl 4-(6-chloro-5-(2-chloroethyl)-2-morpholinopyrimidin-4-ylamino)piperidine-1-carboxylate (100 mg, 0.217 mmol) and sodium iodide (7 mg, 0.047 mmol) in acetonitrile (10 mL) at room temperature. The resultant mixture was refluxed for 4 h under nitrogen atmosphere. After cooling to room temperature, the mixture was diluted with ethyl acetate (80 mL), washed with water (30 mL) and brine (30 mL). The organics were dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography, eluting with petroleum ether/ethyl acetate=9/1 then 3/1 to obtain tert-butyl 4-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate (20 mg, 22%) as white solid. LCMS (ESI) m/z: 424.3 [M+H].sup.+.

Step 3: Synthesis of tert-butyl 4-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate

[0253] A suspension of pyridin-3-ylmethanol (10 mg, 0.092 mmol) and sodium hydride (5 mg, 0.125 mmol) in tetrahydrofuran (3 mL) was stirred at room temperature for 10 min followed by the addition of tert-butyl 4-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate (20 mg, 0.047 mmol). The reaction mixture was then refluxed for 72 h and cooled. It was then diluted with ethyl acetate (80 mL), washed with water (30 mL×2) and brine (20 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain tert-butyl 4-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate (14.6 mg, 62%) as white solid. .sup.1H NMR (400 MHz, MeOD) δ 8.60 (d, J=1.6 Hz, 1H), 8.47 (dd, J=5.2, 1.6 Hz, 1H), 7.90 (dt, J=8.0, 1.6 Hz, 1H), 7.44 (dd, J=8.0, 0.8 Hz, 1H), 5.42 (s, 2H), 4.18 (d, J=124 Hz, 2H), 4.03 (pent, J=7.6 Hz, 11H), 3.68 (s, 8H), 3.54 (t, J=8.4 Hz, 2H), 2.82-2.78 (m, 4H), 1.73-1.68 (m, 4H), 1.48 (s, 9H). LCMS (ESI) m/z: 497.1 [M+H].sup.+.

Step 4: Synthesis of 4-(7-(piperidin-4-yl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0254] Trifluoroacetic acid (1 ml) was added to a solution of tert-butyl 4-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)piperidine-1-carboxylate (60 mg, 0.121 mmol) in dichloromethane (2 mL) at room temperature. After stirring at room temperature for 2 h, the reaction mixture was concentrated. The residue was subjected to prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.0% aqueous ammonium bi(arbonaie) to obtain 4-(7-(piperidin-4-yl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (9.5 mg, 20%) as white solid. .sup.1H NMR (400 MHz, CD3OD) δ 8.60 (s, 1H), 8.48 (d, J=4.4 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.44 (dd, J=8.0, 4.8 Hz, 1H), 5.42 (s, 2H), 4.08-4.03 (m, 1H), 3.69 (s, 8H), 3.58 (t, J=8.0 Hz, 2H), 3.32-3.30 (m, 2H), 2.91-2.80 (m, 4H), 1.90-1.81 (m, 4H). LCMS (ESI)/z: 397.1 [M+H].

[0255] The following compounds were synthesized according to the protocol described above:

TABLE-US-00004 Name Structure NMR, MS # 4-(2-morpholino-4- (pyridin-3-ylmethoxy)- 5H-pyrrolo[2,3- d]pyrimidin-7(6H)- yl)benzonitrile [00163] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.66 (d, J = 1.6 Hz, 1H), 8.53 (dd, J = 4.8, 1.6 Hz, 1H), 7.93 (d, J = 9.2 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.8 Hz, 2H), 7.41 (dd, J = 7.6, 4.8 Hz, 1H), 5.45 (s, 2H), 4.06 (t, J = 8.4 Hz, 2H), 3.67 (s, 8H), 2.92 (t, J = 8.4 Hz, 2H). LCMS (ESI) m/z: 415.2 [M + H].sup.+. 21 4-(7-(3-fluorophenyl)-4- (pyridazin-3-ylmethoxy)- 6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00164] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.18 (t, J = 3.2 Hz, 1H), 7.76 (dt, J = 11.2, 2.4 Hz, 1H), 7.71 (d, J = 3.2 Hz, 2H), 7.50 (dd, J = 8.0, 1.2 Hz, 1H), 7.37 (q, J = 4.4 Hz, 1H), 6.79 (dt, J = 10.4, 2.0 Hz, 1H), 5.658 (s, 2H), 4.05 (t, J = 8.4 Hz, 2H), 3.58 (s, 8H), 2.95 (t, J = 8.8 Hz, 2H); LC-MS: m/z = 409.2 (M + H).sup.+. 14 4-(7-(pyrimidin-5-yl)-4- ((tetrahydro-2H-pyran-4- yl)methoxy)-6,7-dihydro- 5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00165] .sup.1H NMR (400 MHz, Chloroform-d) δ 9.19 (s, 2H), 8.83 (s, 1H), 4.21 (d, J = 6.5 Hz, 2H), 4.03 (t, J = 9.2 Hz, 4H), 3.80 (m, 8H), 3.45 (dt, J = 9.6, 2.0 Hz, 2H), 3.02 (t, J = 9.2, 7.8 Hz, 2H), 2.09-1.96 (m, 1H), 1.74-1.66 (m, 2H), 1.52-1.39 (m, 2H). LCMS (ESI) m/z: 399.3 [M + H].sup.+. 77 4-(4-((1-ethylpiperidin-3- yl)methoxy)-7-(pyridin- 3-yl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00166] .sup.1H NMR (400 MHz, Chloroform-d) δ 9.06 (d, J = 2.7 Hz, 1H), 8.24 (d, J = 4.8 Hz, 1H), 8.08 (dd, J = 9.8, 2.4 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 4.27-4.22 (m, 1H), 4.18-4.10 (m, 1H), 4.03 (t, J = 8.5 Hz, 2H), 3.78 (s, 8H), 3.06-2.91 (m, 4H), 2.49-2.35 (m, 2H), 2.17-2.03 (m, 1H), 1.92-1.84 (m, 1H), 1.83-1.72 (m, 3H), 1.67-1.60 (m, 1H), 1.13-1.00 (m, 4H). LCMS (ESI) m/z: 425.3 [M + H].sup.+. 92 4-(4-(2-(1- methylpiperidin-3- yl)ethoxy)-7-(pyridin-3- yl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00167] .sup.1H NMR (400 MHz, Chloroform-d) δ 9.05 (d, J = 2.7 Hz, 1H), 8.22 (dd, J = 4.4, 1.2 Hz, 1H), 8.06 (dd, J = 11.2, 2.4 Hz, 1H), 7.26-7.22 (m, 1H), 4.36 (t, J = 6.4 Hz, 2H), 4.01 (t, J = 8.6 Hz, 2H), 3.76 (s, 8H), 2.97 (t, J = 9.2 Hz, 2H), 2.90-2.76 (m, 2H), 2.27 (s, 3H), 1.92-1.76 (m, 4H), 1.70-1.57 (m, 4H), 0.97-0.88 (m, 1H). LCMS (ESI) m/z: 425.4 [M + H].sup.+. 93 4-methyl-2-((2- morpholino-7-(pyridin-3- yl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 4- yloxy)methyl)morpholine [00168] .sup.1H NMR (400 MHz, Chloroform-d) δ 9.05 (s, 1H), 8.23 (d, J = 4.6 Hz, 1H), 8.05 (dd, J = 9.6, 1.2 Hz, 1H), 7.28 (d, J = 4.8 Hz, 1H), 4.40 (dd, J = 17.2, 6.0 Hz, 1H), 4.32 (dd, J = 16.4, 5.2 Hz, 1H), 4.01 (t, J = 9.2, 8.0 Hz, 2H), 3.96-3.87 (m, 2H), 3.79-3.67 (m, 9H), 3.01 (t, J = 9.3, 7.9 Hz, 2H), 2.87-2.80 (m, 1H), 2.70-2.63 (m, 1H), 2.32 (s, 3H), 2.21-2.11 (m, 1H), 1.97 (t, J = 11.2, 10.3 Hz, 1H). LCMS (ESI) m/z: 413.3 [M + H].sup.+. 94 4-(7-(3-methoxyphenyl)- 4-((1-methylpiperidin-3- yl)methoxy)-6,7-dihydro- 5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00169] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.67 (s, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 6.56 (dd, J = 8.0, 2.4 Hz, 1H), 4.25 (dd, J = 10.8, 5.6 Hz, 1H), 4.13 (dd, J = 10.8, 7.2 Hz, 1H), 3.99 (t, J = 8.4 Hz, 2H), 3.79 (s, 3H), 3.75 (s, 8H), 3.01-2.99 (m, 2H), 2.91-2.86 (m, 3H), 2.32 (s, 3H), 2.11-2.04 (m, 1H), 2/04-1.99 (m, 1H), 1.78-1.77 (m, 3H), 1.67-1.65 (m, 1H), 1.19-1.06 (m, 1H); LC- MS : m/z = 440.3 (M + H).sup.+. 95 4-(7-phenyl-4- (pyrimidin-5- ylmethoxy)-6,7-dihydro- 5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00170] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.15 (s, 1H), 8.90 (s, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.00 (t, J = 7.2 Hz, 1H), 5.46 (s, 2H), 4.03 (t, J = 8.4 Hz, 2H), 3.65 (s, 8H), 2.91 (t, J = 8.8 Hz, 2H); LC-MS: m/z = 391.2 (M + H).sup.+. 96 4-(7-phenyl-4-(pyrazin- 2-ylmethoxy)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00171] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.75 (s, 1H), 8.62 (s, 1H), 8.56 (s, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.33 (t, J = 8 Hz, 2H), 7.01-6.99 (m, 1H), 5.56 (s, 2H), 4.09 (t, J = 8.0 Hz, 2H), 3.68 (s, 8H), 3.04 (t, J = 8.0 Hz, 2H); LC-MS: m/z = 391.2 (M + H).sup.+. 97 4-(4-((1-methylpiperidin- 3-yl)methoxy)-7- (pyrimidin-5-yl)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00172] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.20 (s, 2H), 8.78 (s, 1H), 4.23-4.04 (m, 4H), 3.66 (s, 8H), 2.93 (t, J = 8.4 Hz, 2H), 2.78-2.62 (m, 2H), 2.16 (s, 3H), 1.98-1.89 (m, 2H), 1.76-1.61 (m, 3H), 1.49 (m, 1H), 1.03 (m, 1H). LCMS (ESI) m/z: 412.1 [M + H].sup.+. 80 4-(4-((1-methylpiperidin- 3-yl)methoxy)-7- (pyridazin-4-yl)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00173] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.77 (d, J = 2.4 Hz, 1H), 8.93 (d, J = 6.0 Hz, 1H), 7.80 (dd, J = 6.0, 2.4 Hz, 1H), 4.24-4.13 (m, 2H), 4.06 (t, J = 8.4 Hz, 2H), 3.34 (s, 8H), 2.93 (t, J = 8.4 Hz, 2H), 2.79-2.63 (m, 2H), 2.17 (s, 3H), 2.00-1.79 (m, 3H), 1.70-1.46 (m, 3H), 1.01 (m, 1H). LCMS (ESI) m/z: 412.3 [M + H].sup.+. 81 4-(7-(1-methyl-1H- pyrazol-4-yl)-4-((1- methylpiperidin-3- yl)methoxy)-6,7-dihydro- 5H-pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00174] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (s, 1H), 7.63 (s, 1H), 4.18-4.07 (m, 2H), 3.84-3.79 (m, 5H), 3.65 (s, 8H), 2.88 (t, J = 8.6 Hz, 2H), 2.77-2.62 (m, 2H), 2.15 (s, 3H), 1.97-1.92 (m, 2H), 1.77-1.59 (m, 3H), 1.47 (m, 1H), 0.98 (m, 1H). LCMS (ESI) m/z: 414.2 [M + H].sup.+. 82 2-methyl-1-(2- morpholino-7-(pyridin-3- yl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 4-yloxy)propan-2-ol [00175] .sup.1H NMR (400 MHz, Chloroform-d) δ 9.05 (s, 1H), 8.25 (s, 1H), 8.08-8.06 (m , 1H), 7.32-7.27 (m, 1H), 4.25 (s, 2H), 4.05 (t, J = 8.5 Hz, 2H), 3.81-3.68 (m, 8H), 3.61 (s, 1H), 3.02 (t, J = 8.5 Hz, 2H), 1.30 (s, 6H). LCMS (ESI) m/z: 372.2 [M + H].sup.+. 83 4-(4-(oxetan-3- ylmethoxy)-7-phenyl- 6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00176] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.75 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 6.97 (t, J = 7.6 Hz, 1H), 4.68 (dd, J = 7.6, 6.0 Hz, 2H), 4.52 (d, J = 6.8 Hz, 2H), 4.40 (t, J = 6.0 Hz, 2H), 4.00 (t, J = 8.4 Hz, 2H), 3.66 (s, 8H), 3.36-3.350 (m, 1H), 2.85 (t, J = 7.2 Hz, 2H); LC-MS: m/z = 369.3 (M + H).sup.+. 99 4-(7-phenyl-4- ((tetrahydro-2H-pyran-4- yl)oxy)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00177] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.75 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 8 Hz, 2H), 6.97 (t, J = 7.2 Hz, 1H), 5.20 (sept, J = 4.4 Hz, 1H), 4.02 (t, J = 8.4 Hz, 2H), 3.88-3.82 (m, 2H), 3.65 (d, J = 4.4 Hz, 8H), 3.53-3.47 (m, 2H), 2.88 (t, J = 8.4 Hz, 2H), 2.01-1.97 (m, 2H), 1.66-1.59 (m, 2H); LCMS (ESI) m/z: 383.1 [M + H].sup.+. 11 4-(7-phenyl-4- ((tetrahydrofuran-3- yl)oxy)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00178] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.75 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8 Hz, 2H), 6.98 (t, J = 7.2 Hz, 1H), 5.52 (sept, J = 2.0 Hz, 1H), 4.02 (t, J = 8.4 Hz, 2H), 3.92 (dd, J = 10.0, 4.8 Hz, 1H), 3.84 (dd, J = 15.6, 8.0 Hz, 1H), 3.77-3.70 (m, 2H), 3.66 (s, 8H), 2.89-2.85 (m, 2H), 2.22 (dd, J = 13.6, 6.8 Hz, 1H), 2.00 (d, J = 6.8 Hz, 1H); LCMS (ESI) m/z: 369.1 [M + H].sup.+. 12

Synthesis of 4-(7-phenyl-4-((pyridin-2-ylmethoxy)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 43)

[0256] ##STR00179##

[0257] To a solution of pyridin-2-ylmethanol (29 mg, 0.27 mmol) in THE (8 mL) was added sodium hydride (11 mg, 0.27 mmol) at 0° C. portion wise. The mixture was stirred at 0° C. for 30 min followed by the addition of (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methyl methanesulfonate (70 mg, 0.18 mmol). The resultant mixture was stirred at 80° C. for 16 h, then quenched with water (10 mL) and extracted with dichloromethane (20 mL*3). The organic layer was washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by prep-HPLC (0.05% NH4HCO3/H2O:CH3CN=5%˜95%) to obtain 4-(7-phenyl-4-((pyridin-2-ylmethoxy)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (1.8 mg, 15%,) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 8.59 (d, J=4.4 Hz, 1H), 7.79 (d, J=8.0 Hz, 2H), 7.74 (dt, J=8.0, 2.0 Hz, 1H), 7.53 (d, J=8.0 Hz, 1H), 7.40 (t, J=7.6 Hz, 2H), 7.25-7.22 (m, 1H), 7.06 (t, J=7.6 Hz, 1H), 4.77 (s, 2H), 4.56 (s, 2H), 4.06 (t, J=8.4 Hz, 2H), 3.79 (s, 8H), 3.16 (t, J=8.4 Hz, 2H); LCMS (ESI) m/z: 404.1 [M+H].sup.+.

[0258] The following compounds were synthesized according to the protocol described above:

TABLE-US-00005 Name Structure NMR, MS # tert-butyl 3-((2- morpholino-7- (pyridin- 3-yl)-6,7- dihydro-5H- pyrrolo[2,3-d] pyrimidin- 4- yl)methoxy) pyrrolidine- 1-carboxylate [00180] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.10 (bs, 1H), 8.29 (bs, 1H), 8.10 (d, J = 8 Hz, 1H), 7.29 (d, J = 3.6 Hz, 1H), 4.41 (s, 2H), 4.16 (bs, 1H), 4.03 (t, J = 8.4 Hz, 2H), 3.76 (s, 8H), 3.57-3.43 (m, 4H), 3.15 (t, J = 8.4 Hz, 2H), 2.08-1.96 (m, 2H), 1.46 (s, 9H). LCMS (ESI) m/z: 483.1 [M + H].sup.+. 15 4-(7-(pyridin- 3-yl)-4- ((pyrrolidin-3- yloxy)methyl)- 5,6- dihydro-5H- pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00181] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.05 (d, J = 2.4 Hz, 1H), 8.24-8.21 (m, 2H), 7.42-7.38 (m, 1H), 4.35 (s, 2H), 4.21 (bs, 1H), 4.07 (t, J = 8.4 Hz, 2H), 3.65 (s, 8H), 3.41-3.29 (m, 2H), 3.12-3.04 (m, 5H), 1.99-1.87 (m, 2H). LCMS (ESI) m/z: 383.1 [M + H].sup.+. 13 4-(4-((oxetan-3- yloxy)methyl)-7- (pyridin-3-yl)- 6,7- dihydro-5H- pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00182] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.05 (d, J = 2.8 Hz, 1H), 8.24-8.20 (m, 2H), 7.42-7.29 (m, 1H), 4.70-4.66 (m, 3H), 4.46-4.44 (m, 2H), 4.30 (s, 2H), 4.08 (t, J = 8.4 Hz, 2H), 3.65 (s, 8H), 3.10 (t, J = 8.4 Hz, 2H). LCMS (ESI) m/z: 370.1 [M + H].sup.+. 31 4-(4-((1- methylpyrrolidin- 3- yloxy)methyl)-7- (pyridin-3-yl)-6,7- dihydro-5H- pyrrolo[2,3- d]pyrimidin-2- yl)morpholine [00183] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.05 (d, J = 2.0 Hz, 1H), 8.24-8.20 (m, 2H), 7.42-7.39 (m, 1H), 4.31 (s, 2H), 4.16 (bs, 1H), 4.07 (t, J = 8.4 Hz, 2H), 3.65 (s, 8H), 3.10 (t, J = 8.4 Hz, 2H), 2.86-2.73 (m, 3H), 2.60-2.55 (m, 1H), 2.40 (s, 3H), 2.12-2.02 (m, 1H), 1.85-1.75 (m, 1H). LCMS (ESI) m/z: 397.1 [M + H].sup.+. 29

Synthesis of 4-(4-methoxy-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 16)

[0259] ##STR00184##

[0260] To a solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.34 mmol) in methanol (80 mL) was added sodium methoxide (8 mL). The mixture was refluxed overnight and concentrated. The crude product obtained was purified by prep-HPLC to give 4-(4-methoxy-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (18.3) mg as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.75 (d, J=6.8 Hz, 2H), 7.34 (t, J=6.0 Hz, 2H), 6.97 (t, J=5.6 Hz, 1H), 4.01 (t, J=6.8 Hz, 2H), 3.85 (s, 3H), 3.67 (s, 8H), 2.86 (t, J=6.8 Hz, 2H); LCMS (ESI) m/z: 313 [M+H].sup.+.

Synthesis of 4-(7-(3-fluorophenyl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 17)

[0261] ##STR00185##

Step 1: Synthesis of 4-(4-chloro-7-(3-fluorophenyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0262] To a solution of 3-fluoroaniline (181 mg, 1.63 mmol) in THE (20 mL) was added NaH (130 mg, 3.25 mmol) at 0° C. slowly. The mixture was stirred at 60° C. for 2 h and then 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (400 mg, 1.35 mmol) was added. The resultant mixture was stirred at 110° C. for 16 h and then quenched with saturated aqueous NH.sub.4Cl solution (20 mL). The mixture was extracted with EtOAc (50*3 mL), the combined organics were washed with brine (100 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by SGC (PE/EA=1:1) to obtain 4-(4-chloro-7-(3-fluorophenyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (270 mg, 59%) as yellow solid. LCMS (ESI) m/z: 335.0 [M+H].sup.+.

Step 2: Synthesis of 4-(7-(3-fluorophenyl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0263] To a solution of pyridin-3-ylmethanol (86 mg, 0.79 mmol) in THE (20 mL) was added NaH (32 mg, 0.79 mmol) at 0° C. slowly. The mixture was stirred at 0° C. for 2 h and then 4-(4-chloro-7-(3-fluorophenyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (220 mg, 0.66 mmol) was added. The resultant mixture was stirred at 110° C. for 16 h and concentrated. The crude product obtained was purified by prep-HPLC (0.05% FA/H2O:CH3CN=5%˜95%) to obtain 4-(7-(3-fluorophenyl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (47.2 mg, 18%,) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.67 (s, 1H), 8.54 (d, J=4.0 Hz, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.77 (d, J=12.8 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.43-7.33 (m, 2H), 6.81-6.76 (m, 1H), 5.45 (s, 2H), 4.03 (t, J=8.8 Hz, 2H), 3.67 (s, 8H), 2.90 (t, J=8.8 Hz, 2H); LCMS (ESI) m/z: 408.1 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-(pyridin-2-yloxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 18) and 1-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyridin-2(1H)-one (Compound 76)

[0264] ##STR00186##

[0265] To a solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.32 mmol) in DMF (10 mL) were added pyridin-2-ol (33 mg, 0.35 mmol) and K.sub.2CO.sub.3 (88 mg, 0.64 mmol) and the resultant mixture was stirred at 140° C. for 16 h. Then the reaction was quenched with water (5 mL) and was extracted with EtOAc (20*3 mL). The organic layer was combined, washed with brine (30 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was purified by prep-HPLC (0.05% NH.sub.4HCO.sub.3/H.sub.2O:CH.sub.3CN=5%˜95%) to offer 4-(7-phenyl-4-(pyridin-2-yloxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (9.3 mg, 8%) and 1-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyridin-2(1H)-one (9.0 mg, 8%) as yellow solids.

[0266] Compound 18: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.31 (dd, J=4.8, 1.2 Hz, 1H), 7.79-7.75 (m, 3H), 7.41-7.36 (m, 2H), 7.15-7.04 (m, 3H), 4.07 (t, J=8.4 Hz, 2H), 3.74-3.69 (m, 8H), 2.92 (t, J=8.4 Hz, 2H); LCMS (ESI) m/z: 376.1 [M+H].sup.+.

[0267] Compound 76: .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.77 (d, J=7.6 Hz, 2H), 7.67 (dd, J=1.6, 6.4 Hz, 1H), 7.43-7.39 (m, 3H), 7.10 (t, J=7.6 Hz, 1H), 7.63 (d, J=9.2 Hz, 1H), 6.28 (t, J=6.4 Hz, 1H), 4.11 (t, J=8.4 Hz, 2H), 3.81-3.77 (m, 8H), 3.05 (t, J=8.4 Hz, 2H); LCMS (ESI) m/z: 376.1 [M+H].sup.+.

[0268] The following compounds were synthesized according to the protocol described above:

TABLE-US-00006 Name Structure NMR, MS # 4-(7-phenyl-4-(pyridin-3- yloxy)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00187] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.48 (d, J = 2.6 Hz, 1H), 8.42 (dd, J = 4.7, 1.3 Hz, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.67 (ddd, J = 8.3, 2.8, 1.4 Hz, 1H), 7.46 (dd, J = 8.3, 4.7 Hz, 1H), 7.43-7.32 (m, 2H), 7.03 (t, J = 7.3 Hz, 1H), 4.11 (t, J = 8.5 Hz, 2H), 3.67-3.54 (m, 4H), 3.47 (s, 4H), 3.00 (t, J = 8.5 Hz, 2H); LCMS (ESI) m/z: 376.2 [M + H].sup.+. 22 4-(7-phenyl-4-(pyridin-4- yloxy)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00188] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.38 (d, J = 7.8 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.47-7.31 (m, 2H), 7.09 (t, J = 7.2 Hz, 1H), 6.54 (d, J = 7.9 Hz, 2H), 4.22- 4.10 (m, 2H), 3.77 (dd, J = 19.2, 5.2 Hz, 8H), 3.26 (d, J = 8.4 Hz, 2H); LCMS (ESI) m/z: 376.0 [M + H].sup.+. 23 4-(7-(pyridin-3-yl)-4-(pyridin- 3-yloxy)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00189] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.03 (d, J = 2.0 Hz, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.43 (dd, J = 3.6, 0.9 Hz, 1H), 8.23 (dd, J = 3.6, 0.8 Hz, 1H), 8.20 (dq, J = 6.8, 1.2 Hz, 1H), 7.67 (dq, J = 6.4, 1.2 Hz, 1H), 7.48 (dd, J = 3.6, 0.8 Hz, 1H), 7.41 (dd, J = 6.8, 1.8 Hz, 1H), 4.15 (t, J = 6.8 Hz, 2H), 3.57 (s, 4H), 3.47 (s, 4H), 3.03 (t, J = 7.2 Hz, 2H); LCMS (ESI) m/z: 377.3 [M + H]+. 24 4-(7-(3-fluorophenyl)-4- (pyridin-3-yloxy)-6,7-dihydro- 5H-pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00190] .sup.1H NMR (400 MHz, CDCL3) δ 8.53 (d, J = 2.0 Hz, 1H), 8.43 (dd, J = 3.8, 0.8 Hz, 1H), 7.75 (dt, J = 6.0, 2.0 Hz, 1H), 7.51 (dq, J = 6.4, 1.2 Hz, 1H), 7.36-7.26 (m, 3H), 6.75-6.71 (m, 1H), 4.08 (t, J = 6.8 Hz, 2H), 3.70- 3.60 (m, 8H), 3.06 (t, J = 7.0 Hz, 2H). LCMS (ESI) m/z: 394.2 [M + H].sup.+. 74

Synthesis of 4-(7-(pyridin-3-yl)-4-((tetrahydrofuran-2-yl)methoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 19)

[0269] ##STR00191##

Step 1: Synthesis of 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0270] A solution of pyridin-3-amine (238 mg, 2.53 mmol) in tetrahydrofuran (15 mL) was added to a suspension of sodium hydride (202 mg, 5.06 mmol) in tetrahydrofuran (10 mL) at 0° C. The reaction mixture was then refluxed for 1 h. After cooling to room temperature, 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (500 mg, 1.69 mmol) was added and the mixture was refluxed further for 16 h. The reaction mixture was poured then into ice water (50 mL) and extracted with ethyl acetate (50 mL×2). The organic layer was washed with brine (40 mL), dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=1/3 to 0/100) to obtain 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (400 mg, 74%). LCMS (ESI) m/z: 318.1 [M+H].sup.+.

Step 2: Synthesis 4-(7-(pyridin-3-yl)-4-((tetrahydrofuran-2-yl)methoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0271] A solution of (tetrahydrofuran-2-yl)methanol (80 mg, 0.78 mmol) in THE (3 mL) was added to a solution of sodium hydride (38 mg, 0.95 mmol) in tetrahydrofuran (5 mL) at 0° C. After stirring at room temperature for 10 min, 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.31 mmol) was added. The resultant reaction mixture was refluxed for 12 h. After cooling, the reaction mixture was diluted with ethyl acetate (80 mL), washed with water (30 mL×2) and brine, dried over sodium sulphate, filtered and concentrated. The residue was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to give 4-(7-(pyridin-3-yl)-4-((tetrahydrofuran-2-yl)methoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine_(28.8 mg, 24%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.99 (d, J=2.4 hz, 1H), 8.18-8.15 (m, 2H), 7.36 (dd, J=8.4, 4.4 Hz, 1H), 4.30-4.22 (m, 2H), 4.20-4.13 (m, 1H), 4.04 (t, J=8.6 hz, 2H), 3.80-3.75 (m, 1H), 2.90 (t, J=8.6 hz, 2H), 1.99-1.81 (m, 3H), 1.70-1.64 (m, 1H). LCMS (ESI) m/z: 384.1 [M+H].sup.+.

Synthesis of 3-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)benzonitrile (Compound 20)

[0272] ##STR00192##

Step 1: Synthesis of 3-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)benzonitrile

[0273] To a suspension of sodium hydride (40 mg, 1.0 mmol) in tetrahydrofuran (10 mL) was added 3-aminobenzonitrile (48 mg, 0.405 mmol) at 0° C. The reaction mixture was then refluxed for 1 h and cooled to room temperature. A solution of 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (100 mg, 0.337 mmol) in THE was added to the mixture and then it was refluxed for another 16 h. After cooling to room temperature, the reaction mixture was quenched with water (50 mL) and the precipitate formed was collected by filtration, washed with methanol and dried to give 3-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)benzonitrile (60 mg, 52%). The crude product was used in next step without further purification. LCMS (ESI) m/z: 342.0 [M+H].sup.+.

Step 2: Synthesis of 3-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)benzonitrile

[0274] Pyridin-3-ylmethanol (48 mg, 0.440 mmol) was added to a suspension of sodium hydride (21 mg, 0.525 mmol) in tetrahydrofuran (10 mL) at room temperature and stirred for 10 min. Then 3-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)benzonitrile (60 mg, 0.176 mmol) was added. The resultant mixture was refluxed for 12 h and cooled. Water (30 mL) was added to the mixture and the solids formed was collected by filtration to afford the crude product. It was then purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 3-(2-morpholino-4-(pyridin-3-ylmethoxy)-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)benzonitrile (11.3 mg, 16%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.67 (s, 1H), 8.53 (d, J=4.8 Hz, 1H), 8.16 (d, J=8 Hz, 1H), 8.12 (s, 1H), 7.85 (d, J=8 Hz, 1H), 7.55 (s, 1H), 7.43-7.39 (m, 2H), 5.45 (s, 2H), 4.06 (t, J=8.6 Hz, 2H), 3.67 (s, 8H), 2.92 (t, J=8.6 Hz, 2H). LCMS (ESI) m/z: 415.0 [M+H].sup.+.

Synthesis of 3-((2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)propane-1,2-diol (Compound 25)

[0275] ##STR00193##

Step 1: Synthesis of 4-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0276] A solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (90 mg, 0.68 mmol) in THF (5 mL) was added to a suspension of sodium hydride (27 mg, 0.68 mmol) in THF (5 mL) at 0° C. The reaction mixture was refluxed for 2 h and cooled. Then a solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.34 mmol) in 3 mL of THE was added and the resultant mixture was stirred at reflux for 16 h. The reaction mixture was then diluted with ethyl acetate (30 mL), the resultant organic medium was washed with brine (10 mL), dried over sodium sulfate and concentrated to obtain 100 mg of the target compound. This was used in the next step without further purification.

Step 2: Synthesis of 3-((2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)propane-1,2-diol

[0277] A solution of 4-(4-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.24 mmol) in water (1 mL) and acetic acid (5 mL) was stirred at 80° C. overnight. The resultant mixture concentrated, and the crude product obtained was purified by prep-HPLC to give title compound 5.2 mg as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.76 (d, J=8.0 Hz, 2H), 7.34 (t, J=7.6 Hz, 2H), 6.97 (t, J=7.6 Hz, 1H), 4.87 (d, J=5.2 Hz 1H), 4.64 (t, J=8.8 Hz, 1H), 4.25-4.30 (m, 1H), 4.00-4.15 (m, 1H), 4.02 (t, J=8.4 Hz, 2H), 3.75-3.80 (m, 1H), 3.67 (s, 8H), 3.40 (t, J=5.6 Hz, 2H), 2.88 (t, J=9.2 Hz, 2H); LCMS (ESI) m/z: 373.0 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-((pyridin-3-yloxy)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 26) and 5-hydroxy-1-((2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methyl)pyridin-1-ium-3-ylium (Compound 71)

[0278] ##STR00194##

Step 1: Synthesis of methyl 2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-4-carboxylate

[0279] A solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (500 mg, 1.578 mmol), triethylamine (479 mg, 4.734 mmol), palladium(II) acetate (36 mg, 0.160 mmol) and 1.1′-bis(diphenylphosphino)ferrocene (131 mg, 0.236 mmol) in methanol (12 mL) and dimethyl sulfoxide (15 mL) was stirred at 80° C. for 16 h under carbon monoxide atmosphere. After cooling to room temperature, the reaction mixture was filtered through celite, the filtrate was diluted with ethyl acetate (150 mL) and washed with water (40 mL×3) and brine (30 mL). The organics were dried over sodium sulfate, filtered, concentrated and the crude product obtained was purified by silica gel column chromatography, eluting with PE/EA=3/1 to obtain methyl 2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-4-carboxylate (400 mg, 74%) as yellow solid. LCMS (ESI) m/z: 341.1 [M+H].sup.+.

Step 2: Synthesis of (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methanol

[0280] Lithium aluminum hydride (1.76 mL, 1.76 mmol) was added in portions to a solution of methyl 2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-4-carboxylate (400 mg, 1.175 mmol) in tetrahydrofuran (20 mL) at 0° C. The solution was stirred at 0° C. for 1 h, then quenched with sodium sulfate decahydrate (2 g) and filtered through celite and washed with dichloromethane. The filtrate was concentrated to give (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methanol (170 mg, 46%) as white solid. LCMS (ESI) m/z: 313.1 [M+H].sup.+. This crude product was used in the next step without further purification.

Step 3: Synthesis of Compound 26 and Compound 71

[0281] To a solution of triphenylphosphine (118 mg, 0.450 mmol), pyridin-3-ol (43 mg, 0.452 mmol) and (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methanol (70 mg, 0.224 mmol) in tetrahydrofuran (15 mL) was added DIAD (91 mg, 0.450 mmol) at room temperature. The resultant mixture was stirred at room temperature for 1 h and concentrated. The residue was subjected to prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain compound 26 (17.7 mg, 20%) and compound 71 (12 mg, 14%) as white solids.

[0282] Compound 26: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.36 (d, J=2.8 Hz, 1H), 8.20-8.18 (m, 1H), 7.81 (d, J=8 Hz, 2H), 7.45-7.32 (m, 4H), 7.03 (t, J=7.6 Hz, 1H), 5.03 (s, 2H), 4.05 (t, J=8.4 Hz, 2H), 3.65 (s, 8H), 3.05 (t, J=8.4 Hz, 2H). LCMS (ESI) m/z: 390.1 [M+H].sup.+.

[0283] Compound 71: .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ7.79 (d, J=6.0 Hz, 2H), 7.46-7.43 (m, 2H), 7.38 (t, J=6.0 Hz, 2H), 7.29 (dd, J=7.2, 4.4 Hz, 1H), 7.05 (t, J=6.0 Hz, 1H), 6.97-6.94 (m, 1H), 5.23 (s, 2H), 4.09 (t, J=6.6 Hz, 2H), 3.59 (dd, J=12.0, 3.6 Hz, 8H), 3.00 (t, J=6.6 Hz, 2H). LCMS (ESI) m/z: 390.1 [M].

Synthesis of 4-(1-phenyl-6-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine (Compound 27)

[0284] ##STR00195##

Step 1: Synthesis of 4,6-dichloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine

[0285] To a stirred solution of 2,4,6-trichloropyrimidine-5-carbaldehyde (630 mg, 3 mmol) in ethanol (20 mL) were added phenyl hydrazine (324 mg, 3 mmol) and triethylamine (910 mg, 9 mmol) dropwise at −78° C. in that order. The resultant mixture was stirred at −78° C. for 0.5 h and at 0° C. for 2 h. The mixture was then quenched with water (20 mL), the resultant precipitate was collected by filtration and dried to give 4,6-dichloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine as white solid. (790 mg, 99%). LCMS (ESI) m/z: 265.0 [M+H].sup.+.

Step 2: Synthesis of 4-(6-chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine

[0286] To a mixture of 4,6-dichloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (792 mg, 3 mmol) in dichloromethane (10 mL) were added morpholine (520 mg, 6 mmol) and DIPEA (774 mg, 6 mmol) at 0° C. and the resultant mixture was stirred at room temperature for 16 h. The mixture was concentrated and purified by column chromatography eluting with 0-30% ethyl acetate in petroleum ether to give 4-(6-chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine as a yellow solid. (800 mg, 85%). LCMS (ESI) m/z: 316.0 [M+H].sup.+.

Step 3: Synthesis 4-(1-phenyl-6-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine

[0287] To a mixture of pyridin-3-ylmethanol (218 mg, 2 mmol) in tetrahydrofuran (10 mL) was added sodium hydride (120 mg, 3 mmol) at 0° C. followed by 4-(6-chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine (315 mg, 1 mmol) and the resultant mixture was stirred at room temperature for 16 h. The reaction was quenched with water (10 mL) and the formed precipitate was collected by filtration and dried.

[0288] The crude product thus obtained was purified with Prep-HPLC (BOSTON pHlex ODS 10 um 21.2×250 mm120 A. The mobile phase was acetonitrile/0.1% Formic acid) to obtain 4-(1-phenyl-6-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)morpholine as yellow solid. (75 mg, 19%). .sup.1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J=1.6 Hz, 1H), 8.54 (dd, J=4.8, 1.6 Hz, 1H), 8.47 (s, 1H), 8.11 (dd, J=4.8, 0.8 Hz, 2H), 7.90-7.87 (m, 1H), 7.56-7.52 (m, 2H), 7.43-7.32 (m, 2H), 5.44 (s, 2H), 3.92 (t, J=4.8 Hz, 4H), 3.75 (t, J=4.8 Hz, 4H); LCMS (ESI) m/z: 389.2 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-(((tetrahydro-2H-pyran-4-yl)oxy)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 30)

[0289] ##STR00196##

Step 1: Synthesis of (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methyl methanesulfonate

[0290] Methanesulfonyl chloride (37 mg, 0.33 mmol) was added to a solution of (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methanol (70 mg, 0.22 mmol) and triethylamine (44 mg, 0.44 mmol) in dichloromethane (8 mL) at 0° C. The reaction mixture was stirred at 0° C. for 1 h under nitrogen atmosphere and then quenched with saturated aqueous sodium bicarbonate solution (10 mL) and extracted with dichloromethane (20 mL*3). The organic layer was washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated to give (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methyl methanesulfonate (70 mg, 72%) as brown solid. The crude product was used in the next step without further purification. LCMS (ESI) m/z: 391.0 [M+H].sup.+.

Step 2: Synthesis of 4-(7-phenyl-4-(((tetrahydro-2H-pyran-4-yl)oxy)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0291] A suspension of tetrahydro-2H-pyran-4-ol (27 mg, 0.27 mmol) and sodium hydride (11 mg, 0.27 mmol) in tetrahydrofuran (10 mL) was stirred at room temperature for 30 min, followed by the addition of (2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)methylmethane sulfonate (70 mg, 0.18 mmol) to the mixture. Then resultant mixture was stirred at 80° C. for 16 h, then quenched with water (10 mL) and extracted with dichloromethane (20*3 mL). The organic layer was washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated. The residue was subjected to prep-HPLC (0.05% NH.sub.4HCO.sub.3/H.sub.2O:CH.sub.3CN=5%˜95%) to offer 4-(7-phenyl-4-(((tetrahydro-2H-pyran-4-yl)oxy)methyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (4.3 mg, 6%,) as yellow solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 7.79 (d, J=7.6 Hz, 2H), 7.41-7.37 (m, 2H), 7.07 (t, J=7.6 Hz, 1H), 4.46 (s, 2H), 4.06 (t, J=8.4 Hz, 2H), 4.01-3.96 (m, 2H), 3.78 (s, 8H), 3.69-3.62 (m, 1H), 3.53-3.47 (m, 2H), 3.17 (t, J=8.4 hz, 2H), 2.01-1.97 (m, 2H), 1.71-1.67 (m, 2H); LCMS (ESI) m/z: 397.2 [M+H].sup.+.

Synthesis of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 32)

[0292] ##STR00197##

[0293] Step 1: Synthesis of 5-allylpyrimidine-2,4,6(1H,3H,5H)-trione.

[0294] To a solution of diethyl 2-allylmalonate (40.0 g, 200.0 mmol) and urea (12.0 g, 200.0 mmol) in ethanol (150 mL) was added sodium ethoxide (20% in ethanol) (80 mL) and the mixture was heated to 85° C. for 3 h. The resultant mixture was cooled to 20° C. and acetone (150 mL) was added. After stirring for 10 min and resultant precipitate was collected by filtration, washed with petroleum ether (150 mL) and then dissolved into water (150 mL). The pH of the resultant solution was adjusted between 3-4 with conc. HCl to obtain a precipitate which was stirred for 10 min. The solids were collected by filtration and dried under high vacuum to obtain 5-allylpyrimidine-2,4,6(1H,3H,5H)-trione as a brown solid (17.0 g, 51%). .sup.1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 2H), 5.63-5.73 (m, 1H), 5.03 (dd, J=12.0 Hz, J=3.6 Hz, 2H), 3.68 (t, J=5.2 Hz, 1H), 2.66 (t, J=5.62 Hz, 2H); LCMS (ESI) m/z: 169.1 [M+H].sup.+.

Step 2: Synthesis of 5-allyl-2,4,6-trichloropyrimidine

[0295] To a solution of 5-allylpyrimidine-2,4,6(1H,3H,5H)-trione (17.0 g, 101.2 mmol) in phosphorus oxychloride (60 mL) was added N,N-dimethylaniline (8.5 mL) and the solution was heated to 110° C. for 4 h. The dark solution was then cooled to 20° C. and concentrated. Ethyl acetate (300 mL) and ice water (200 mL) were added to the residue, the organic phase was separated and washed with brine (200 mL), dried, concentrated to obtain the crude product. It was purified by column chromatography (petroleum ether:ethyl acetate from 20:1 to 10:1) to obtain 5-allyl-2,4,6-trichloropyrimidine as off-white solid (16.0 g, 71%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.79-5.89 (m, 1H), 5.11-5.21 (m, 2H), 3.63 (dt, J=6.0 Hz, J=1.6 Hz, 2H); LCMS (ESI) m/z: 223.1 [M+H].sup.+.

Step 3: Synthesis of 2-(2,4,6-trichloropyrimidin-5-yl)acetaldehyde

[0296] To a solution of 5-allyl-2,4,6-trichloropyrimidine (10.0 g, 44.7 mmol), potassium osmate(VI) dihydrate (330 mg, 0.89 mmol) and 4-methylmorpholine N-oxide (20.96 g, 89.4 mmol) in acetone (150 mL) and water (150 mL) was added sodium periodate (38.3 g, 178.8 mmol) at 0° C. and the mixture was stirred at 0-20° C. for 17 h. The resultant mixture was filtered and the filtrate was concentrated to remove the acetone and the aqueous phase was extracted with ethyl acetate (150 mL×2). The combined organic layer was washed with brine (150 mL), dried, concentrated to obtain the crude product. It was then purified by silica gel chromatography (petroleum ether:acetic ester from 10:1 to 3:1) to obtain 2-(2,4,6-trichloropyrimidin-5-yl)acetaldehyde as grey solid (5.9 g, 59%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.80 (s, 1H), 4.14 (s, 2H).

Step 4: Synthesis of 2,4-dichloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine

[0297] To a solution of 2-(2,4,6-trichloropyrimidin-5-yl)acetaldehyde (2.2 g, 9.76 mmol) and aniline (1.09 g, 11.71 mmol) in methanol (60 mL) were added acetic acid (1.0 mL) and sodium cyanoborohydride (1.23 g, 19.52 mmol) at 0° C. The resultant mixture was stirred between 0-20° C. for 17 h. Water (60 mL) was added to the mixture and after 10 mins the resultant precipitate was collected by filtration and dried under vacuum to obtain 2,4-dichloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine as white solid (2.0 g, 77%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.69 (dd, J=8.8 Hz, J=1.2 Hz, 2H), 7.39-7.44 (m, 2H), 7.16 (t, J=7.2 Hz, 1H), 4.21 (t, J=8.8 Hz, 2H), 3.17 (t, J=8.8 Hz, 2H); LCMS (ESI) m/z: 266.1 [M+H].sup.+.

Step 5: Synthesis of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0298] A solution of 2,4-dichloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine (100 mg, 0.376 mmol) and morpholine (164 mg, 1.88 mmol) in tetrahydrofuran (10 mL) was heated to 50° C. for 17 h. The mixture was concentrated to dryness followed by the addition of acetonitrile (5 mL) and water (20 mL) to the residue. The resultant precipitate was collected by filtration and dried under vacuum to obtain 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine as white solid (67 mg, 56%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.78 (d, J=7.6 Hz, 2H), 7.39 (t, J=6.8 Hz, J=2.0 Hz, 2H), 7.06 (t, J=7.2 Hz, 1H), 4.10 (t, J=8.8 Hz, 2H), 3.65 (s, 8H), 2.99 (t, J=8.8 Hz, 2H); LCMS (ESI) m/z: 317.1 [M+H].sup.+.

Synthesis of 4-(4-chloro-7-(3-methylbenzyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 33)

[0299] ##STR00198##

[0300] A mixture of 2,4-dichloro-7-(3-methylbenzyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine (2.0 g, 6.80 mmol) and morpholine (2.96 g, 34.0 mmol) in tetrahydrofuran (40 mL) was heated to 35° C. for 17 h and concentrated to dryness. MeOH (40 mL) and water (40 mL) were added to the residue and stirred. The resultant precipitate was collected by filtration and dried in vacuum to obtain 4-(4-chloro-7-(3-methylbenzyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (2.0 g, 85%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.23 (t, J=8.0 Hz, 2H), 7.05-7.10 (m, 3H), 4.48 (s, 2H), 3.61 (s, 8H), 3.48 (t, J=8.4 Hz, 2H), 2.85 (t, J=8.4 Hz, 2H), 2.29 (s, 3H); LCMS (ESI) m/z: 345.1 [M+H].sup.+.

Synthesis of 4-(7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 34)

[0301] ##STR00199##

Step 1a: Synthesis of morpholine-4-carboximidamide hydrochloride

[0302] N,N-Diisopropylethylamine (2.58 g, 20.00 mmol) was added to a solution of morpholine (1.74 g, 20.00 mmol) and 1H-pyrazole-1-carboximidamide hydrochloride (2.92 g, 20.00 mmol) in N,N-dimethylformamide (5 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 h and ethyl ether (50 mL) was added to the mixture. The oily product at the bottom of the flask was solidified by repeated sonication and fresh ethyl ether. The solid was then collected by filtration and dried to obtain morpholine-4-carboximidamide hydrochloride (3 g, 91%) as white solid. LCMS (ESI) m/z: 130.1 [M+H].sup.+.

Step 1: Synthesis of methyl 2-oxotetrahydrofuran-3-carboxylate

[0303] A solution of dihydrofuran-2(3H)-one (3.36 g, 39.02 mmol) in tetrahydrofuran (5 mL) was added dropwise to lithium hexamethyldisilazide (1.0 M in tetrahydrofuran, 80.0 mL, 80.0 mmol) at −78° C. After stirring at −78° C. for 10 min, dimethyl carbonate (3.69 g, 40.98 mmol) was added at the same temperature. The reaction mixture was warmed up and stirred at room temperature for 16 h. Then it was poured onto a mixture of concentrated hydrochloric acid (15 mL) and ice (150 mL), followed by extraction with ethyl acetate (200 mL×2). The organic layer was washed by brine, dried over sodium sulfate and concentrated to obtain methyl 2-oxotetrahydrofuran-3-carboxylate (4.9 g, 87%). LCMS (ESI) m/z: 144.9 [M+H].sup.+.

Step 2: Synthesis of 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine

[0304] Morpholine-4-carboximidamide hydrochloride (575 mg, 3.47 mmol) was added to a solution of methyl 2-oxotetrahydrofuran-3-carboxylate (500 mg, 3.47 mmol) and sodium methoxide (287 mg, 5.31 mmol) in methanol (5 mL) at room temperature. The reaction mixture was refluxed for 2 h and concentrated. The resulting residue was dissolved in phosphorus oxychloride (5 mL) and heated with stirring at 100° C. for 16 h. Then the reaction mixture was added dropwise to water (100 mL), and then neutralized with 5 M aqueous sodium hydroxide solution. It was extracted with ethyl acetate (50 mL×2), the combined organic layer was washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography (n-hexane/ethyl acetate=10/1) to obtain 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (236 mg, 23%) as white solid. LCMS (ESI) m/z: 298.0 [M+H].sup.+.

Step 3: Synthesis of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0305] A solution of aniline (157 mg, 1.69 mmol) in tetrahydrofuran (3 mL) was added to a solution of sodium hydride (68 mg, 1.70 mmol) in tetrahydrofuran (2 mL) at 0° C. The reaction mixture was then refluxed for 2 h and cooled. Then 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (100 mg, 0.34 mmol) was added at room temperature and the resultant mixture was refluxed for 16 h. It was cooled, then poured into ice water (30 mL) and extracted with ethyl acetate (20 mL×2). The organic layer was washed with brine (20 mL), dried over sodium sulfate and concentrated. The crude product obtained was purified by silica gel column chromatography (petroleum ether/ethyl acetate=9/1) to obtain 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (90 mg, 82%). LCMS (ESI) m/z: 317.0 [M+H].sup.+.

Step 4: Synthesis 4-(7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0306] A suspension of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (80 mg, 0.25 mmol) and Pd/C (30 mg) in methanol (10 mL) and ethyl acetate (2 mL) was stirred at room temperature for 30 min under hydrogen atmosphere. The reaction solution was filtered through celite and the filtrated was concentrated. The crude product obtained was purified by PREP-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate) to afford 4-(7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (49.2 mg, 70%) as light-yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.85 (s, 1H), 7.80 (d, J=8 Hz, 2H), 7.37 (t, J=8 Hz, 2H), 7.02 (t, J=7.6 Hz, 1H), 4.04 (t, J=8.4 Hz, 2H), 3.64 (s, 8H), 2.99 (t, J=8.4 Hz, 2H). LCMS (ESI) m/z: 283.1 [M+H].sup.+.

Synthesis of 4-(4-methyl-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 35) and 2-methyl-1-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)propan-2-ol (Compound 84)

[0307] ##STR00200##

Step 1: Synthesis of 4-(4-Methyl-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0308] A mixture of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (50 mg, 0.158 mmol), 2,4,6-trimethyl-1,3,5,2,4,6-trioxatriborinane (40 mg, 0.316 mmol), tris(dibenzylideneacetone)dipalladium (15 mg, 0.016 mmol), tris(dibenzylideneacetone)dipalladium (9 mg, 0.032 mmol) and cesium carbonate (103 mg, 0.316 mmol) in dimethyl sulfoxide (2 mL) and water (0.5 mL) was stirred at 140° C. for 16 h under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (80 mL), washed with water (40 mL×3), brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography, eluting with petroleum ether/ethyl acetate=6/1 to obtain 4-(4-methyl-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (31.6 mg, 68%). .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.80 (d, J=7.5 Hz, 2H), 7.36 (dd, J=8.5, 7.5 Hz, 2H), 7.00 (t, J=7.5 Hz, 1H), 4.03 (t, J=8.5 Hz, 2H), 3.64 (s, 8H), 2.95 (t, J=8.5 Hz, 2H), 2.13 (s, 3H). LCMS (ESI) m/z: 297.2 [M+H].sup.+.

Step 2: Synthesis of 2-methyl-1-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)propan-2-ol

[0309] To a stirred solution of 4-(4-methyl-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.338 mmol) in tetrahydrofuran (5 mL) at 0° C. was added n-butyl lithium (0.25 mL, 0.506 mmol) and the resultant mixture was stirred at 0° C. for 0.5 h. Then propan-2-one (29 mg, 0.101 mmol) was added and the mixture was stirred further at room temperature for 2 h. Then water (20 mL) was added and the mixture was extracted with ethyl acetate (30 mL×3). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by prep-HPLC (Column Xbridge 21.2*250 mm C18, 10 um, mobile phase A: water (10 mmol/L ammonium bicarbonate) B: acetonitrile) to obtain 2-methyl-1-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)propan-2-ol (35.7 mg, 30%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.81 (d, J=8.4 Hz, 2H), 7.37 (t, J=7.6 Hz, 2H), 7.01 (t, J=6.4 Hz, 1H), 5.01 (s, 1H), 4.03 (t, J=8 Hz, 2H), 3.66-3.60 (m, 8H), 3.00 (t, J=8.4 Hz, 2H), 2.53 (s, 2H), 1.17 (s, 6H); LC-MS: m/z=355.2 (M+H).sup.+.

Synthesis of 2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-4-carbonitrile (Compound 36)

[0310] ##STR00201##

[0311] A mixture of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.316 mmol), zinc cyanide (74 mg, 0.631 mmol) and bis(tri-tert-butylphosphine)palladium(0) (32 mg, 0.063 mmol) in N,N-dimethylacetamide (4 mL) in a sealed vial was heated with microwave irradiation at 150° C. for 0.5 h under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (80 mL), washed with water (40 mL×3) and brine (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The resultant crude product was purified by silica gel column chromatography, eluting with petroleum ether/ethyl acetate=6/1 to give 2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-4-carbonitrile (33.0 mg, 34%) as yellow solid. 1H NMR (400 MHz, DMSO-d.sub.6) δ 7.81 (d, J=8.0 Hz, 2H), 7.42 (t, J=8 Hz, 2H), 7.12 (s, 1H), 4.16 (t, J=8.0 Hz, 2H), 3.65 (s, 8H), 3.16 (t, J=8.0 Hz, 2H). LCMS (ESI) m/z: 308.1 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-(pyridin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 37)

[0312] ##STR00202##

[0313] A solution of 2-methylpyridine (64 mg, 0.7 mmol) in tetrahydrofuran (15 mL) was added to n-BuLi (1 mL, 2.5 mmol, 2.5 M solution in hexanes) at 0° C. and stirred for 1 h. Then a solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 0.64 mmol) in THE was added and the resultant mixture was warmed up to room temperature and stirred for 16 h. Then the reaction was quenched with saturated aqueous NH.sub.4Cl solution (10 mL) and extracted with EtOAc (15*3 mL). The organic layer was combined, washed with brine (30 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by prep-HPLC (0.05% FA/H.sub.2O:CH.sub.3CN=5%˜95%) to afford 4-(7-phenyl-4-(pyridin-2-ylmethyl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (39.3 mg, 17%,) as white solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J=4.0 Hz, 1H), 7.80 (d, J=8.0 Hz, 2H), 7.74-7.00 (m, 1H), 7.39-7.33 (m, 3H), 7.25-7.22 (m, 1H), 7.01 (t, J=7.2 Hz, 1H), 4.02 (t, J=8.4 Hz, 2H), 3.95 (s, 2H), 3.63 (s, 8H), 2.92 (t, J=8.4 Hz, 2H); LCMS (ESI) m/z: 374.3 [M+H].sup.+.

[0314] The following compound was synthesized according to the protocol described above:

TABLE-US-00007 Name Structure NMR, MS # 4-(4-(pyridin- 2-ylmethyl)- 7-(pyridin-3- yl)-6,7- dihydro-5H- pyrrolo[2,3- d]pyrimidin- 2-yl) morpholine [00203] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.08 (d, J = 3.0 Hz, 1H), 8.54 (d, J = 5.6 Hz, 1H), 8.28 (dd, J = 4.8, 1.2 Hz, 1H), 8.13 (ddd, J = 8.4, 2.8, 1.2 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.36-7.28 (m, 2H), 7.18-7.15 (m, 1H), 4.06 (s, 2H), 4.02 (t, J = 8.8 Hz, 2H), 3.00 (t, J = 8.0 Hz, 2H); LCMS (ESI) m/z: 375.3 [M + H]+. 38

Synthesis of 4-(7-phenyl-4-(pyridin-2-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 39)

[0315] ##STR00204##

[0316] A solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (250 mg, 0.79 mmol), pyridin-2-ylboronic acid (486 mg, 3.95 mmol), 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (131 mg, 0.16 mmol) and cesium carbonate (772 mg, 2.37 mmol) in water (5.0 mL) and DMSO (20 mL) was stirred at 130° C. for 8 h under argon atmosphere.

[0317] The mixture was diluted with ethyl acetate (150 mL), washed with water (150 mL) and the organic layer was concentrated. The crude product obtained was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%-95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 4-(7-phenyl-4-(pyridin-2-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine as yellow solid (44.3 mg, 15%). .sup.1H NMR (400 MHz, Chloroform-d) δ 8.67 (d, J=4.9, 1.7 Hz, 1H), 8.37 (d, J=8.0 Hz, 1H), 7.90-7.72 (m, 3H), 7.42 (t, J=8.0 Hz, 2H), 7.30-7.27 (m, 1H), 7.06 (t, J=7.4 Hz, 1H), 4.12 (t, J=8.3 Hz, 2H), 3.94-3.75 (m, 8H), 3.60 (t, J=8.3 Hz, 2H). LCMS (ESI) m/z: 360.2 [M+H].sup.+.

[0318] The following compounds were synthesized according to the above protocol.

TABLE-US-00008 Name Structure NMR, MS # 4-(7-phenyl-4- (pyridin-3-yl)- 6,7-dihydro- 5H-pyrrolo [2,3-d] pyrimidin-2- yl) morpholine [00205] .sup.1H NMR (500 MHz, Chloroform-d) δ 9.12 (d, J = 2.2, 0.9 Hz, 1H), 8.64 (dd, J = 4.8, 1.7 Hz, 1H), 8.28 (dt, J = 8.0, 2.0 Hz, 1H), 7.81 (d, J = 8 Hz, 2H), 7.44-7.35 (m, 3H), 7.10 (t, J = 7.2 Hz, 1H), 4.12 (t, J = 8.2 Hz, 2H), 3.91-3.75 (m, 8H), 3.35 (t, J = 8.2 Hz, 2H). LCMS (ESI) m/z: 360.1 [M + H].sup.+. 40 4-(7-phenyl-4- (pyridin-4-yl)- 6,7-dihydro- 5H-pyrrolo [2,3-d] pyrimidin-2- yl) morpholine [00206] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.74 (dd, J = 4.0, 1.2 Hz, 1H), 7.82-7.77 (m, 4H), 7.43 (t, J = 6.4 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H), 4.13 (t, J = 8.2 Hz, 2H), 3.90- 3.84 (m, 4H), 3.83-3.77 (m, 4H), 3.34 (t, J = 8.2 Hz, 2H). LCMS (ESI) m/z: 360.1 [M + H].sup.+. 41

Synthesis of 4-(7-(1-methylpiperidin-4-yl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 44)

[0319] ##STR00207##

Step 1: Synthesis of 6-chloro-5-(2-chloroethyl)-N-(1-methylpiperidin-4-yl)-2-morpholinopyrimidin-4-amine

[0320] To a stirred solution of 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (100 mg, 0.337 mmol) and 1-methylpiperidin-4-amine (38 mg, 0.333 mmol) in acetonitrile (10 mL) at room temperature was added N-ethyl-N-isopropylpropan-2-amine (109 mg, 0.843 mmol). The reaction mixture was then refluxed for 16 h and cooled. It was diluted with ethyl acetate (80 mL), washed with water (20 mL), brine (20 mL), dried over sodium sulfate, filtered and concentrated to obtain 6-chloro-5-(2-chloroethyl)-N-(1-methylpiperidin-4-yl)-2-morpholinopyrimidin-4-amine (100 mg, 79%) as white solid. LCMS (ESI) m/z: 374.0 [M+H].sup.+.

Step 2: Synthesis of 4-(4-chloro-7-(1-methylpiperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0321] Cesium carbonate (218 mg, 0.669 mmol) was added to a solution of 6-chloro-5-(2-chloroethyl)-N-(1-methylpiperidin-4-yl)-2-morpholinopyrimidin-4-amine (100 mg, 0.267 mmol) and sodium iodide (8 mg, 0.053 mmol) in acetonitrile (20 mL) at room temperature. The resultant mixture was refluxed for 4 h under nitrogen and cooled. It was diluted with ethyl acetate (150 mL), washed with water (50 mL) and brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography, eluting with dichloromethane/methanol=9/1 to give 4-(4-chloro-7-(1-methylpiperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (30 mg, 0.089 mmol, 33%) as white solid. LCMS (ESI) m/z: 338.1 [M+H].sup.+.

Step 3: Synthesis of 4-(7-(1-methylpiperidin-4-yl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0322] To a suspension of sodium hydride (9 mg, 0.225 mmol) in tetrahydrofuran (5 mL) was added pyridin-3-ylmethanol (20 mg, 0.183 mmol) at room temperature and stirred for 10 min. Then a solution of 4-(4-chloro-7-(1-methylpiperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (30 mg, 0.089 mmol) in THF was added to the mixture and the resultant mixture was refluxed for 48 h. It was cooled, diluted with ethyl acetate (80 mL), washed with water (30 mL) and brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 4-(7-(1-methylpiperidin-4-yl)-4-(pyridin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (14.5 mg, 40%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.61 (d, J=1.6 Hz, 1H), 8.50 (dd, J=4.8, 1.2 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.39 (dd, J=7.6, 4.8 Hz, 1H), 5.35 (s, 2H), 3.74-3.70 (m, 1H), 3.59 (s, 8H), 3.48 (t, J=8.4 Hz, 2H), 2.82-2.79 (m, 2H), 2.71 (t, J=8.4 Hz, 2H), 2.15 (s, 3H), 1.94-1.89 (m, 2H), 1.74-1.68 (m, 2H), 1.56-1.53 (m, 2H). LCMS (ESI) m/z: 411.3 [M+H].sup.+.

Synthesis of 4-(1-phenyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)morpholine (Compound 45)

[0323] ##STR00208##

Step 1: Synthesis of 6-chloro-1-phenyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine

[0324] To a solution of pyridin-3-ylmethanol (109 mg, 1 mmol) in tetrahydrofuran (10 mL) was added sodium hydride (60 mg, 1.5 mmol, 60%) at 0° C. followed by 4,6-dichloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (264 mg, 1 mmol) and the resultant mixture was stirred at room temperature for 16 h. The reaction was quenched with water (10 mL) and the mixture was extracted with ethyl acetate (20 mL*2). The organic layer was dried and concentrated to give 6-chloro-1-phenyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine as yellow solid. (250 mg, 74%). LCMS (ESI) m/z: 338.0 [M+H].sup.+.

Step 2: Synthesis 4-(1-phenyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)morpholine

[0325] To a mixture of 6-chloro-1-phenyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidine (170 mg, 0.5 mmol) in dichloromethane (10 mL) was added morpholine (82 mg, 1 mmol) at 0° C. followed by DIPEA (129 mg, 1 mmol) and the resultant mixture was stirred at room temperature for 16 h. It was then concentrated and the obtained crude product was purified by prep-HPLC (BOSTON pHlex ODS 10 um 21.2×250 mm120 A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to obtain 4-(1-phenyl-4-(pyridin-3-ylmethoxy)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)morpholine as yellow solid. (40 mg, 21%). .sup.1H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J=4.6 Hz, 1H), 8.58 (dd, J=4.8 Hz, 1H), 8.20-8.18 (m, 3H), 7.95 (d, J=8 Hz, 1H), 7.53 (t, J=8 Hz, 2H), 7.45 (dd, J=7.6, 4.8 hz, 1H), 7.30 (t, J=6.8 Hz, 1H), 5.63 (s, 2H), 3.83 (t, J=4.4 Hz, 4H), 3.70 (t, J=4.8 Hz, 4H); LCMS (ESI) m/z: 389.1 [M+H]+.

Synthesis of 2-morpholino-8-phenyl-4-(pyridin-3-ylmethoxy)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one (Compound 46)

[0326] ##STR00209##

[0327] Step 1: Synthesis of methyl 2-(6-hydroxy-2-morpholino-4-oxo-1,4-dihydropyrimidin-5-yl)acetate.

[0328] To a solution of triethyl ethane-1,1,2-tricarboxylate (3 g, 12.18 mmol) and morpholine-4-carboximidamidehydrochloride (2 g, 12.18 mmol) in methanol (40 mL) was added sodium methanolate (30% solution in methanol, 6.7 mL, 34.35 mmol). After the addition, the mixture was stirred at 80° C. for 17 h and concentrated. The crude product methyl 2-(6-hydroxy-2-morpholino-4-oxo-1,4-dihydropyrimidin-5-yl)acetate (3 g, 91.56%) obtained as brown solid was used in the next step without further purification. LCMS (ESI) m/z: 270.0 [M+H].sup.+.

Step 2: Synthesis of methyl 2-(4,6-dichloro-2-morpholinopyrimidin-5-yl)acetate

[0329] A mixture of methyl 2-(6-hydroxy-2-morpholino-4-oxo-1,4-dihydropyrimidin-5-yl)acetate (3 g, 11.15 mmol) and phosphorus oxychloride (20 mL) was stirred at 110° C. for 16 h and then concentrated. The residue was diluted with ethyl acetate/water (20 mL/20 mL), organic layer separated and the aqueous layer was extracted with ethyl acetate (20 mL) twice. The combined organic phase was washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by Combi-Flash (Biotage, 40 g silicagel, eluted with ethyl acetate in petro ether from 10% to 30%) to afford methyl 2-(4,6-dichloro-2-morpholinopyrimidin-5-yl)acetate (1.3 g, 38.2%) as white solid. LCMS (ESI) m/z: 306.1 [M+H].sup.+.

Step 3: Synthesis of methyl 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yl)acetate

[0330] To a solution of pyridin-3-ylmethanol (0.18 g, 1.65 mmol) in tetrahydrofuran (10 mL) was added sodium hydride (100 mg, 2.5 mmol) in portions and the mixture was stirred at 20° C. for 10 min. Then a solution of methyl 2-(4,6-dichloro-2-morpholinopyrimidin-5-yl)acetate (0.5 g, 1.64 mmol) in tetrahydrofuran (2 mL) was added slowly. After the addition, the mixture was stirred at 20° C. for 2 h, then quenched with water (15 mL) and extracted with ethyl acetate (20 mL). The organic phase was dried over sodium sulfate, filtered and concentrated. The residue was purified by Combi-Flash (Biotage, 40 g silica gel, eluted with ethyl acetate in petroleum ether from 30% to 40%) to afford methyl 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yl)acetate (0.3 g, 48.4%) as white solid. LCMS (ESI) m/z: 379.2 [M+H].sup.+.

Step 4: Synthesis of 2-morpholino-4-(pyridin-3-ylmethoxy)-7-m-tolyl-5H-pyrrolo[2,3-d]pyrimidin-6(7H)-one

[0331] A mixture of methyl 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yl)acetate (0.16 g, 0.42 mmol), tris(dibenzylideneacetone)dipalladium (39 mg, 0.042 mmol), 2-(Dicyclohexylphosphino)-2′,4′,6′-triisopropylbiphenyl (40 mg, 0.084 mmol) and cesium carbonate (0.34 g, 1.06 mmol) in toluene (15 mL) was stirred at 90° C. for 3 h under nitrogen atmosphere. The reaction mixture was filtered and concentrated. The residue was purified by prep-HPLC to afford 2-morpholino-4-(pyridin-3-ylmethoxy)-7-m-tolyl-5H-pyrrolo[2,3-d]pyrimidin-6(7H)-one (48 mg, 27.4%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.69 (d, J=1.6 Hz, 1H), 8.55 (dd, J=4.8, 1.2 hz, 1H), 7.88 (d, J=8 Hz, 1H), 7.46-7.34 (m, 2H), 7.27-7.16 (m, 3H), 5.49 (s, 2H), 3.64-3.51 (m, 10H), 2.35 (s, 3H); LCMS (ESI) m/z: 417.9 [M+H].sup.+.

Synthesis of 2-morpholino-8-phenyl-4-(pyridin-3-ylmethoxy)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one (Compound 47)

[0332] ##STR00210##

Step 1: Synthesis of dimethyl 2-(2-ethoxy-2-oxoethoxy)malonate

[0333] A mixture of 1,3-dimethoxy-1,3-dioxopropane-2-diazonium (4 g, 25 mmol), ethyl 2-hydroxyacetate (1.2 mL, 12.5 mmol), and rhodium (II) acetate diner (2 g, 4.5 mmol) in dichloromethane (40 mL) was stirred at 25° C. for 16 h. The reaction mixture was diluted with dichloromethane (20 mL) and filtered. The filtrate was concentrated and the residue was purified by flash chromatography (Biotage, 40 g silica gel, eluted with ethyl acetate in petro ether from 30% to 60%) to afford dimethyl 2-(2-ethoxy-2-oxoethoxy)malonate (3.3 g, 56%) as colorless oil. LCMS (ESI) m/z: 235.1 [M+H].sup.+.

Step 2: Synthesis of methyl 2-(6-hydroxy-2-morpholino-4-oxo-1,4-dihydropyrimidin-5-yloxy)acetate

[0334] To a solution of dimethyl 2-(2-ethoxy-2-oxoethoxy)malonate (3 g, 12.82 mmol) and morpholine-4-carboximidamide hydrochloride (2.1 g, 12.82 mmol) in methanol (70 mL) was added sodium methanolate (30% solution in methanol, 7.5 mL, 38.46 mmol). After the addition, the mixture was stirred at 80° C. for 17 h and concentrated to afford methyl 2-(6-hydroxy-2-morpholino-4-oxo-1,4-dihydropyrimidin-5-yloxy)acetate (2.1 g, 57.5%) as brown solid, which was used directly in next step without further purification. LCMS (ESI) m/z: 286.1 [M+H].sup.+.

Step 3: Synthesis of methyl 2-(4,6-dichloro-2-morpholinopyrimidin-5-yloxy)acetate

[0335] A mixture of methyl 2-(6-hydroxy-2-morpholino-4-oxo-1,4-dihydropyrimidin-5-yloxy)acetate (2 g, 7 mmol), N,N-dimethylaniline (0.85 g, 7 mmol) and phosphorus oxychloride (15 mL) was stirred at 110° C. for 16 h. It was concentrated and the residue was diluted with ethyl acetate/water (20 mL/20 mL), the organic layer separated and the aqueous phase was extracted with ethyl acetate (20 mL) twice. The combined organic phase was washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The residue was subjected to flask chromatography (Biotage, 40 g silicagel, eluted with ethyl acetate in petro ether from 10% to 30%) to obtain methyl 2-(4,6-dichloro-2-morpholinopyrimidin-5-yloxy)acetate (0.85 g, 37.8%) as yellow solid. LCMS (ESI) m/z: 322.1 [M+H].sup.+.

Step 4: Synthesis of 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yloxy)acetic acid

[0336] To a solution of pyridin-3-ylmethanol (68 mg, 0.62 mmol) in tetrahydrofuran (10 mL) was added sodium hydride (38 mg, 0.93 mmol) in portions and the mixture was stirred at 20° C. for 10 min. Then a solution of methyl 2-(4,6-dichloro-2-morpholinopyrimidin-5-yloxy)acetate (0.2 g, 0.62 mmol) in tetrahydrofuran (2 mL) was added slowly and the resultant mixture was stirred at 20° C. for 2 h. It was then quenched with water (15 mL) and extracted with ethyl acetate (20 mL). The aqueous phase was lyophilized to afford crude 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yloxy)acetic acid (0.2 g, 87%) as white solid, which was used directly in next step without further purification. LCMS (ESI) m/z: 381.1 [M+H].sup.+.

Step 5: Synthesis of 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yloxy)-N-phenylacetamide

[0337] To a solution of 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yloxy)acetic acid (0.18 g, 0.47 mmol) and aniline (66 mg, 0.71 mmol) in N,N-dimethylformamide (15 mL) was added 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.32 g, 0.85 mmol) in portions, followed by N,N-diisopropylethylamine (0.18 g, 1.42 mmol). The resultant mixture was stirred at 20° C. for 2 h and then diluted with ethyl acetate/water (30 mL, 1:1). The layers were separated and the aqueous phase was extracted with ethyl acetate (20 mL) twice. The combined organic phase was washed with brine (20 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography (Biotage, 20 g silicagel, eluted with 7N ammonia in methanol:dichloromethane=1:10 in dichloromethane from 15% to 20%) to afford 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yloxy)-N-phenylacetamide (0.11 g, 51%) as yellow oil. LCMS (ESI) m/z: 456.1 [M+H].sup.+.

Step 6: Synthesis of 2-morpholino-8-phenyl-4-(pyridin-3-ylmethoxy)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one

[0338] A mixture of 2-(4-chloro-2-morpholino-6-(pyridin-3-ylmethoxy)pyrimidin-5-yloxy)-N-phenylacetamide (0.1 g, 0.22 mmol), tris(dibenzylideneacetone)dipalladium (20 mg, 0.022 mmol), 2-(dicyclohexylphosphino)-2′,4′,6′-triisopropylbiphenyl (21 mg, 0.044 mmol) and cesium carbonate (0.18 g, 0.55 mmol) in toluene (10 mL) was stirred at 100° C. for 16 h under nitrogen atmosphere. It was filtered and concentrated and the residue was subjected to prep-HPLC to afford 2-morpholino-8-phenyl-4-(pyridin-3-ylmethoxy)-6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one (5 mg, 5.4%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.69 (s, 1H), 8.56 (d, J=4.8 Hz, 1H), 7.89 (d, J=8 Hz, 1H), 7.52-7.38 (m, 4H), 7.28 (d, J=7.2 Hz, 2H), 5.46 (s, 2H), 4.77 (s, 2H), 3.54-3.57 (m, 4H), 3.34-3.27 (m, 4H); LCMS (ESI) m/z: 420.0 [M+H].sup.+.

Synthesis of 2-morpholino-N-(oxetan-3-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-amine (Compound 48)

[0339] ##STR00211##

[0340] To a solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (120 mg, 0.38 mmol) in dioxane (10 mL) were added oxetan-3-amine (55 mg, 0.76 mmol), Pd.sub.2(dba).sub.3 (52 mg, 0.057 mmol), Xantphos (66 mg, 0.11 mmol) and Cs.sub.2CO.sub.3 (371 mg, 1.14 mmol). The resultant mixture was stirred at 110° C. for 16 h and concentrated. The crude product obtained was purified by prep-HPLC (0.05% FA/H2O:CH3CN=5%˜95%) to obtain 2-morpholino-N-(oxetan-3-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-amine (14.3 mg, 11%,) as yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.47 (s, 1H), 7.67 (d, J=8.0 Hz, 2H), 7.40 (t, J=7.6 Hz, 2H), 7.11 (t, J=7.6 Hz, 1H), 4.44 (t, J=9.6 Hz, 1H), 4.21 (t, J=8.4 Hz, 3H), 4.08 (dd, J=10.4, 5.6 Hz, 1H), 3.73 (t, J=4.4 Hz, 4H), 3.57-3.45 (m, 6H), 2.95-2.91 (m, 2H); LCMS (ESI) m/z: 354.1 [M+H].sup.+.

[0341] The following compound were synthesized according to the protocol described above:

TABLE-US-00009 Name Structure NMR, MS # 2- morpholino- 7- phenyl-N- (pyridin-3- yl)-6,7- dihydro-5H- pyrrolo [2,3-d] pyrimidin- 4-amine [00212] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.74 (d, J = 2.4 Hz, 1H), 8.28 (dd, J = 4.8, 1.2 Hz, 1H), 7.98-7.95 (m, 1H), 7.74 (dd, J = 8.8, 1.2 Hz, 2H), 7.40-7.36 (m, 2H), 7.25-7.23 (m, 1H), 7.03 (t, J = 7.2 Hz, 1H), 5.94 (s, 1H), 4.11 (t, J = 8.4 Hz, 2H), 3.79 (s, 8H), 2.94 (t, J = 8.4 Hz, 2H); LCMS (ESI) m/z: 375.2 [M + H]+. 52

Synthesis of 4-(7-phenyl-4-(tetrahydro-2H-pyran-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 49)

[0342] ##STR00213##

Step 1: Synthesis of 4-(4-(3,6-dihydro-2H-pyran-4-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0343] A mixture of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (100 mg, 0.32 mmol), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (199 mg, 0.95 mmol), Cs.sub.2CO.sub.3 (309 mg, 0.95 mmol), Pd.sub.2(dba).sub.3 (29 mg, 0.03 mmol) and P(Cy).sub.3 (17 mg, 0.06 mmol) in DMSO (10 mL)/H2O (2 mL) was stirred at 140° C. for 16 h under nitrogen atmosphere. Then the reaction was quenched with water (10 mL) and the mixture was extracted with EtOAc (20*3 mL). The organic layers were combined, washed with brine (30 mL), dried over Na2SO4, filtered and concentrated. The residue was subjected to prep-TLC (PE/EA=4:1) to obtain 4-(4-(3,6-dihydro-2H-pyran-4-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (70 mg, 60%) as yellow solid. LCMS (ESI) m/z: 365.2 [M+H].sup.+.

Step 2: Synthesis of 4-(7-phenyl-4-(tetrahydro-2H-pyran-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0344] A suspension of 4-(4-(3,6-dihydro-2H-pyran-4-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (50 mg, 0.14 mmol) and 10% Pd/C (5 mg) in MeOH (10 mL) was stirred at room temperature for 1 h under hydrogen atmosphere. The mixture was then filtered, concentrated and subjected to prep-HPLC (0.05% FA/H2O:CH3CN=5%˜95%) to afford 4-(7-phenyl-4-(tetrahydro-2H-pyran-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (26.6 mg, 52%) as yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J=8.0 Hz, 2H), 7.37 (t, J=8 Hz, 2H), 7.01 (t, J=7.2 Hz, 1H), 4.04 (t, J=8.4 Hz, 2H), 3.93 (dd, J=11.6, 3.2 Hz, 2H), 3.66 (s, 8H), 3.43 (t, J=10.8 Hz, 2H), 3.02 (t, J=8.4 Hz, 2H), 2.75 (t, J=11.6 Hz, 1H), 1.87-1.83 (m, 2H), 1.57 (d, J=11.2 Hz, 2H); LCMS (ESI) m/z: 367.2 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-(pyridazin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 50)

[0345] ##STR00214##

[0346] A solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (60 mg, 0.19 mmol), 4-(tributylstannyl)pyridazine (70 mg, 0.19 mmol), LiCl (8 mg, 0.19 mmol) and Pd(PPh.sub.3).sub.4 (22 mg, 0.019 mmol) in dioxane (5 mL) was stirred at 100° C. for 16 h under nitrogen atmosphere. It was concentrated and the residue was subjected to prep-HPLC (0.05% NH.sub.4HCO3/H.sub.2O:CH.sub.3CN=5%˜95%) to obtain 4-(7-phenyl-4-(pyridazin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (1.8 mg, 3%,) as yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.74 (d, J=0.8 Hz, 1H), 9.33 (dd, J=5.2, 1.2 Hz, 1H), 8.00 (dd, J=5.2, 2.4 Hz, 1H), 7.81 (d, J=8.0 Hz, 2H), 7.44 (t, J=8.0 Hz, 2H), 7.14 (t, J=7.6 Hz, 1H), 4.20 (t, J=8.0 Hz, 2H), 3.90-3.82 (m, 8H), 3.42 (t, J=8.0 Hz, 2H); LCMS (ESI) m/z: 361.2 [M+H].sup.+.

TABLE-US-00010 Name Structure NMR, MS # 4-(7-phenyl-4-(pyrimidin- 5-yl)-6,7-dihydro-5H-pyrrolo [2,3-d]pyrimidin-2-yl) morpholine [00215] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.32 (s, 2H), 9.28 (s, 1H), 7.86 (d, J = 7.6 Hz, 2H), 7.42 (t, J = 8.4 Hz, 2H), 7.08 (t, J = 7.6 Hz, 1H), 4.15 (t, J = 7.6 Hz, 2H), 3.76-3.69 (m, 8H), 3.41-3.37 (m, 2H); LCMS (ESI) m/z: 361.1 [M + H]+. 51 4-(4-(oxazol-5-yl)-7- phenyl-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00216] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.03 (s, 1H), 7.82 (dd, J = 8.8, 0.8 Hz, 2H), 7.67 (s, 1H), 7.42 (dt, J = 7.6, 2.0 Hz, 2H), 7.10 (t, J = 7.2 Hz, 1H), 4.17 (t, J = 8.0 Hz, 2H), 3.87-3.81 (m, 8H), 3.34 (t, J = 8.0 Hz, 2H); LCMS (ESI) m/z: 350.2 [M + H]+. 51 4-(7-(benzo[d][1,3]dioxol- 5-yl)-4-(pyridin-3-yl)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-2-yl)morpholine [00217] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.10 (d, J = 2.0 Hz, 1H), 8.64 (dd, J = 4.8, 1.2 Hz, 1H), 8.28 (d, J = 8 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.53 (dd, J = 8.4, 4.8 Hz, 1H), 7.11 (dd, J = 8.4, 2.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.02 (s, 2H), 4.06 (t, J = 8.0 Hz, 2H), 3.73-3.67 (m, 8H), 3.31 (t, J = 8.0 Hz, 2H). LCMS (ESI) m/z: 404.2 [M + H].sup.+. 63 4-(7-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)-4- (pyridin-3-yl)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-2-yl)morpholine [00218] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.10 (d, J = 1.8 Hz, 1H), 8.63 (dd, J = 4.8, 1.5 Hz, 1H), 8.25 (dt, J = 7.9, 1.8 Hz, 1H), 7.42 (d, J = 2.6 Hz, 1H), 7.39 (dd, J = 7.9, 4.8 Hz, 1H), 7.22 (dd, J = 8.9, 2.6 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 4.28 (dd, J = 10.3, 5.3 Hz, 4H), 4.05 (t, J = 8.2 Hz, 2H), 3.91-3.84 (m, 4H), 3.82- 3.74 (m, 4H), 3.31 (t, J = 8.2 Hz, 2H); LCMS (ESI) m/z 481.2 [M + H].sup.+. 64 4-(7-(3-fluorophenyl)-4- (pyridin-4-yl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2-yl) morpholine [00219] .sup.1H NMR (400 MHz, CDCL3) δ 8.73 (dd, J = 4.4, 1.6 Hz, 2H), 7.83-7.77 (m, 3H), 7.43-7.37 (m, 2H), 6.81-6.76 (m, 1H), 4.11 (t, J = 8.2 Hz, 2H), 3.81-3.89 (m, 8H), 3.36 (t, J = 8.2 Hz, 2H). LCMS (ESI) m/z: 378.1 [M + H].sup.+. 72 4-(7-(pyridin-3-yl)-4- (pyridin-4-yl)-6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin- 2-yl)morpholine [00220] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.13 (d, J = 2.8 Hz, 1H), 8.73 (dd, J = 4.8, 1.6 Hz, 2H), 8.33 (dd, J = 4.8, 1.6 Hz, 1H), 8.14 (dq, J = 8.4, 1.2 Hz, 1H), 7.79 (dd, J = 4.8, 2.0 Hz, 2H), 7.33 (dd, J = 8.0, 4.0 Hz, 1H), 4.14 (t, J = 8 Hz, 2H), 3.88-3.80 (m, 8H), 3.40 (t, J = 8.8 Hz, 2H); LCMS (ESI) m/z: 361.2 [M + H].sup.+. 73 4-(4,7-di(pyridin-3-yl)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-2-yl)morpholine [00221] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.13-9.12 (m, 2H), 8.65 (dd, J = 7.6, 1.2 Hz, 1H), 8.32 (dd, J = 3.6, 0.8 Hz, 1H), 8.26 (dt, J = 6.4, 1.6 Hz, 1H), 8.14 (dq, J = 6.8, 1.2 Hz, 1H), 7.41 (dd, J = 6.4, 3.6 Hz, 1H), 7.33 (dd, J = 6.8, 4.0 Hz, 1H), 4.13 (t, J = 6.4 Hz, 2H), 3.88-3.80 (m, 8H), 3.39 (t, J = 6.8 Hz, 2H). LCMS (ESI) m/z: 361.2 [M + H].sup.+. 75

Synthesis of tert-butyl 3-(((2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)methyl)pyrrolidine-1-carboxylate (Compound 102), 4-(7-(pyridin-3-yl)-4-(pyrrolidin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 55) and 4-(4-((1-methylpyrrolidin-3-yl)methoxy)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 104)

[0347] ##STR00222##

Step 1: Synthesis of tert-butyl 3-(((2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)methyl)pyrrolidine-1-carboxylate

[0348] To a solution of tert-butyl 3-(hydroxymethyl)pyrrolidine-1-carboxylate (84 mg, 0.42 mmol) in THE (15 mL) was added NaH (30 mg, 0.76 mmol) at 0° C. cautiously. The mixture was stirred at room temperature for 15 min and then 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (120 mg, 0.38 mmol) was added. The resultant mixture was stirred further at 100° C. for 16 h. It was quenched with water (10 mL) and extracted with EA (30*3 mL). The organic layer was washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by SGC (PE/EA=1:1 to 0:1) to obtain tert-butyl 3-(((2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)methyl)pyrrolidine-1-carboxylate (135 mg, 74%) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 9.05 (d, J=2.4 Hz, 1H), 8.23 (d, J=4.0 Hz, 1H), 8.06 (d, J=9.2 Hz, 1H), 7.28 (s, 1H), 4.35-4.27 (m, 2H), 4.04 (t, J=8.4 Hz, 2H), 3.62 (s, 8H), 3.60-3.34 (m, 3H), 3.22-3.16 (m, 1H), 3.03-2.97 (m, 2H), 2.69-2.64 (m, 1H), 2.10-2.04 (s, 1H), 1.82-1.74 (m, 1H), 1.49 (s, 9H); LCMS (ESI) m/z: 483.3 [M+H].sup.+.

Step 2: Synthesis of 4-(7-(pyridin-3-yl)-4-(pyrrolidin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0349] To a solution of tert-butyl 3-(((2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)methyl)pyrrolidine-1-carboxylate (120 mg, 0.25 mmol) in DCM (5 mL) was added TFA (1 mL) at 0° C. The mixture was stirred at room temperature for 2 h and concentrated. The resultant residue was purified by prep-HPLC (0.05% NH.sub.4HCO.sub.3/H.sub.2O:CH3CN=5%˜95%) to offer 4-(7-(pyridin-3-yl)-4-(pyrrolidin-3-ylmethoxy)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (16.8 mg, 63%,) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 9.06 (d, J=2.4 Hz, 1H), 8.24 (d, J=4.4 Hz, 1H), 8.10 (d, J=10.0 Hz, 1H), 7.28 (s, 1H), 4.31 (dd, J=10.8, 6.0 Hz, 1H), 4.23 (dd, J=10.8, 8.0 Hz, 1H), 4.04 (t, J=8.4 Hz, 2H), 3.78 (s, 8H), 3.13-3.10 (m, 1H), 3.08-2.93 (m, 4H), 2.82-2.77 (m, 1H), 2.60-2.53 (m, 1H), 2.01-1.95 (m, 1H), 1.60-1.53 (m, 1H); LCMS (ESI) m/z: 383.1 [M+H].sup.+.

Step 3: Synthesis of 4-(4-((l-methylpyrrolidin-3-yl)methoxy)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0350] To a solution of 4-(7-(pyridin-3-yl)-4-(pyrrolidin-3-ylmethoxy)-6,7-dihydro-.H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (30 mg, 0.076 mmol) in methanol (5 mL) was added formaldehyde (2.5 mg, 0.083 mmol). The mixture was stirred at room temperature for 2 h followed by the addition of sodium cyanoborohydride (24 mg, 0.38 mmol) to the mixture. It was then stirred at room temperature for 12 h. The reaction was then quenched with water (5 mL) and extracted with EA (20*3 mL). The organic layer was washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by prep-HPLC (0.05% NH.sub.4HCO.sub.3/H.sub.2O:CH.sub.3CN=5%˜95%) to obtain 4-(4-((1-methylpyrrolidin-3-yl)methoxy)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (8.7 mg, 28%) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 9.06 (d, J=2.4 Hz, 1H), 8.25 (d, J=3.6 Hz, 1H), 8.09 (d, J=9.6 Hz, 1H), 7.28 (s, 1H), 4.33-4.23 (min, 2H), 4.04 (t, J=8.4 hz, 2H), 3.78 (m, 8H), 3.00 (t, J=8.4 hz, 2H), 2.85-2.83 (n, 1H), 2.75-2.64 (m, 3H), 2.53-2.49 (m, 1H), 2.45 (1, 3H), 2.13-2.03 (m, 4H), 1.87-1.66 (n, 18H); LCMS (ESI) m/z: 397.2 [M+H].sup.+.

[0351] The following compounds were synthesized according to the protocol described above:

TABLE-US-00011 Name Structure NMR, MS # 1-(3-(((2-morpholino-7- (pyridin-3-yl)-6,7- dihydro-5H-pyrrolo[2,3- d]pyrimidin-4-yl)oxy) methyl)pyrrolidin-1-yl) ethan-1-one [00223] .sup.1H NMR (400 MHz, CDCl3) δ 9.08 (d, J = 2.4 Hz, 1H), 8.26-8.25 (m, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.31-7.28 (m, 1H), 4.40-4.26 (m, 2H), 4.08-4.02 (m, 2H), 3.78 (s, 8H), 3.73-3.47 (m, 3H), 3.37- 3.32 (m, 1H), 3.00 (dd, J = 16.8, 8.0 Hz, 2H), 2.80-2.67 (m, 1H), 2.20-2.06 (m, 4H), 1.94-1.89 (m, 1H); LCMS (ESI) m/z: 425.3 [M + H]+. 107 tert-butyl 3-(((2-morpholino- 7-(pyridin-3-yl)-6,7- dihydro-5H-pyrrolo[2,3-d] pyrimidin-4-yl)oxy)methyl) azetidine-1-carboxylate [00224] .sup.1H NMR (400 MHz, CDCl3) δ 9.06 (d, J = 2.4 Hz, 1H), 8.25 (d, J = 4.0 Hz, 1H), 8.08 (dd, J = 8.4, 1.2 Hz, 1H), 7.30-7.28 (m, 1H), 4.47 (d, J = 6.8 Hz, 2H), 4.09-4.01 (m, 4H), 3.82-3.78 (m, 10 H), 2.99 (t, J = 8.4 Hz, 3H), 1.46 (s, 9H); LCMS (ESI) m/z: 469.2 [M + H]+. 106 4-(4-(azetidin-3-ylmethoxy)- 7-(pyridin-3-yl)-6,7- dihydro-5H-pyrrolo[2,3-d] pyrimidin-2-yl)morpholine [00225] .sup.1H NMR (400 MHz, CDCl3) δ 9.05 (d, J = 2.8 Hz, 1H), 8.24 (d, J = 4.0 Hz, 1H), 8.09 (d, J = 9.2 Hz, 1H), 7.30-7.28 (m, 1H), 4.50 (d, J = 6.4 Hz, 2H), 4.04 (t, J = 8.4 Hz, 2H), 3.86 (t, J = 8.4 Hz, 2H), 3.78 (s, 8H), 3.69 (t, J = 7.6 Hz, 2H), 3.26-3.20 (m, 1H), 3.01 (t, J = 8.4 Hz, 2H); LCMS (ESI) m/z: 369.1 [M + H]+.  56 4-(4-((1- methylazetidin-3-yl) methoxy)-7-(pyridin-3-yl)- 6,7-dihydro-5H- pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00226] .sup.1H NMR (400 MHz, CDCl3) δ 9.06 (d, J = 1.2 Hz, 1H), 8.25 (d, J = 4.0 Hz, 1H), 8.11-8.08 (m, 1H), 7.30-7.29 (m, 1H), 4.46 (d, J = 6.8 Hz, 2H), 4.04 (t, J = 8.4 Hz, 2H), 3.75 (s, 8H), 3.46 (t, J = 7.6 Hz, 2H), 3.11 (t, J = 6.8 Hz, 2H), 3.01 (t, J = 8.4 Hz, 2H), 2.89 (hept, J = 6.8 Hz, 1H), 2.36 (s, 3H); LCMS (ESI) m/z: 383.3 [M + H]+. 108

Synthesis of tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate (Compound 100), 4-(4-(piperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 57) and 4-(4-(1-methylpiperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 58)

[0352] ##STR00227##

Step 1: Synthesis of tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-5,6-dihydropyridine-1(2H)-carboxylate

[0353] A mixture of 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (50 mg, 0.157 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (97 mg, 0.314 mmol), tris(dibenzylideneacetone)dipalladium (15 mg, 0.016 mmol), tricyclohexylphosphine (9 mg, 0.032 mmol) and cesium carbonate (103 mg, 0.316 mmol) in acetonitrile (8 mL) and water (2 mL) was refluxed for 16 h under nitrogen atmosphere. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (80 mL), washed with water (30 mL) and brine (20 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography, eluting with petroleum ether/ethyl acetate=1/1 then 0/100 to obtain tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-5,6-dihydropyridine-1(2H)-carboxylate (50 mg, 68%) as brown solid. LCMS (ESI) m/z: 465.3 [M+H].sup.+.

Step 2: Synthesis of tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate

[0354] A suspension of tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-5,6-dihydropyridine-1(2H)-carboxylate (50 mg, 0.108 mmol) and palladium on activated charcoal (10%, 30 mg) in methanol (5 mL) and ethyl acetate (5 mL) was stirred at room temperature for 5 h under hydrogen atmosphere. The resultant mixture was filtered through celite and concentrated. The crude product obtained was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate (4.9 mg, 10%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.03 (d, J=2.4 Hz, 1H), 8.22-8.19 (m, 2H), 7.40-7.37 (m, 1H), 4.09-4.03 (m, 4H), 3.65 (s, 8H), 3.05 (t, J=8.4 Hz, 2H), 2.89-2.67 (m, 3H), 1.67-1.61 (m, 4H), 1.41 (s, 9H). LCMS (ESI) m/z: 467.2 [M+H].sup.+.

Step 3: Synthesis of 4-(4-(piperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0355] Trifluoroacetic acid (1 mL) was added to a solution of tert-butyl 4-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate (50 mg, 0.107 mmol) in dichloromethane (2 mL) at room temperature. After stirring the mixture at room temperature for 2 h, it was concentrated and the resultant residue was subjected to prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%-95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 4-(4-(piperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (12.3 mg, 31%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.04 (d, J=2.4 Hz, 1H), 8.22-8.19 (m, 2H), 7.40-7.37 (m, 1H), 4.01 (t, J=8.4 Hz, 2H), 3.67 (s, 8H), 3.14-3.11 (m, 2H), 3.04 (t, J=8.4 Hz, 2H), 2.74-2.66 (m, 3H), 1.83-1.64 (m, 4H). LCMS (ESI) m/z: 367.3 [M+H].sup.+.

Step 4: Synthesis of 4-(4-(1-methylpiperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0356] Acetic acid (16 mg, 0.266 mmol) was added to a mixture of 4-(4-(piperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (50 mg, 0.136 mmol) and formaldehyde solution (37 wt. % in water) (1 mL) in methanol (10 mL) at room temperature. After stirring at room temperature for 2 h, sodium cyanoborohydride (17 mg, 0.271 mmol) was added to the mixture and stirred further for another 2 h. It was then concentrated and the residue obtained was subjected to prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%-95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 4-(4-(1-methylpiperidin-4-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (34.7 mg, 67%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.04 (s, 1H), 8.21-8.20 (m, 2H), 7.40-7.37 (m, 1H), 4.06 (t, J=8.2 Hz, 2H), 3.66 (s, 9H), 3.03 (t, J=8.2 Hz, 2H), 2.85-2.82 (m, 2H), 2.45-2.41 (m, 1H), 2.17 (s, 3H), 1.94-1.84 (m, 4H), 1.63-1.60 (m, 2H). LCMS (ESI) m/z: 381.3 [M+H].sup.+.

Synthesis of tert-butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)azetidine-1-carboxylate (Compound 101)

[0357] ##STR00228##

[0358] A solution of (1-(tert-butoxycarbonyl)azetidin-3-yl)zinc(II) iodide (0.5M in N,N-dimethylacetamide) (1.264 mL, 0.632 mmol) was added to a solution of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (50 mg, 0.158 mmol) and bis(tri-tert-butylphosphine)palladium(0) (16 mg, 0.031 mmol) in N,N-dimethylacetamide (2 mL) at room temperature. The resultant mixture was stirred at 80° C. for 16 h and then quenched with saturated ammonium chloride solution (10 mL). The mixture was then extracted with ethyl acetate (20 mL×3), the combined organic layers were washed with water (20 mL×2), brine (20 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column.

[0359] The elution system used was a gradient of 5%-95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain tert-butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)azetidine-1-carboxylate (4.7 mg, 7%) as white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.82 (d, J=8.0 Hz, 2H), 7.36 (t, J=7.6 Hz, 2H), 7.03 (t, J=7.2 Hz, 1H), 4.21-4.17 (m, 4H), 4.08 (t, J=8.4 Hz, 2H), 3.80-3.73 (2, 9H), 3.00 (t, J=8.4 Hz, 2H), 1.48 (s, 9H). LCMS (ESI) m/z: 438.1 [M+H]e.

[0360] The following compounds were synthesized according to the protocol described above:

TABLE-US-00012 Name Structure NMR, MS # 4-(4- (piperidin-3- (pyridin- 3-yl)-7- (pyridin-3- yl)-6,7-dihydro- 5H-pyrrolo[2,3- d]pyrimidin- 2-yl)morpholine [00229] .sup.1H NMR (400 MHz, CDCl3) δ 9.08 (d, J = 2.8 Hz, 1H), 8.28 (d, J = 3.6 Hz, 1H), 8.15 (dd, J = 8.4 Hz, 1.2 Hz, 1H), 7.32-7.28 (m, 1H), 4.06 (t, J = 8.4 Hz, 2H), 3.29 (s, 8H), 3.11-2.98 (m, 5H), 2.77-2.67 (m, 2H), 1.90-1.85 (m, 2H), 1.64-1.59 (m, 2H). LCMS (ESI) m/z: 367.0 [M + H]+.  59 4-(4- (azetidin-3- yl)-7-phenyl- 6,7-dihydro- 5H-pyrrolo [2,3-d] pyrimidin- 2-yl) morpholine [00230] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.02 (t, J = 7.2 Hz, 1H), 4.05-4.00 (m, 4H), 3.89-3.84 (m, 1H), 3.80-3.39 (m, 10H), 2.92 (t, J = 8.0 Hz, 2H). LCMS (ESI) m/z: 338.3 [M + H].sup.+.  61 4-(7-phenyl- 4-(pyrrolidin-3- yl)-6,7- dihydro-5H- pyrrolo[2,3-d] pyrimidin- 2-yl) morpholine [00231] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.01 (t, J = 6.8 Hz, 1H), 4.05 (t, J = 8.8 Hz, 2H), 3.65 (s, 8H), 3.49-3.43 (m, 2H), 3.23-3.16 (m, 2H), 3.06-2.96 (m, 4H), 2.07-1.93 (m, 2H); LC-MS: m/z = 352 (M + H).sup.+.  62 4-(4-(1- methylazetidin- 3- yl)-7-phenyl- 6,7-dihydro-5H- pyrrolo[2,3-d] pyrimidin-2- yl)morpholine [00232] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 8.8 Hz, 2H), 7.01 (t, J = 5.6 Hz, 1H), 4.02 (t, J = 8.4 Hz, 2H), 3.68-3.57 (m, 11H), 3.46-3.30 (m, 2H), 2.92 (t, J = 8.4 Hz, 2H), 2.29 (s, 3H). LCMS (ESI) m/z: 352.1 [M + H].sup.+. 103

Synthesis of 8-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)octahydropyrazino[2,1-c][1,4]oxazine (Compound 78)

[0361] ##STR00233##

[0362] A mixture of octahydropyrazino[2,1-c][1,4]oxazine hydrochloride (60 mg, 0.337 mmol), 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (107 mg, 0.337 mmol) and cesium carbonate (328 mg, 1.011 mmol) in N,N-dimethylformamide (5 mL) was stirred at 85° C. for 4 h. Water (10 mL) was then added and the mixture was extracted ethyl acetate (20 mL×3). The organic layer was dried and concentrated. The crude product obtained was purified by SGC (dichloromethane:methanol from 50:1 to 10:1) to obtain 8-(2-morpholino-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)octahydropyrazino[2,1-c][1,4]oxazine (23.2 mg, 16%) as yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 8.97 (d, J=2.8 Hz, 1H), 8.15-8.10 (m, 2H), 7.34 (dd, J=7.6, 4.4 Hz, 1H), 4.26 (d, J=14 Hz, 1H), 4.09 (d, J=12.4 Hz, 1H), 3.94 (t, J=8.4 Hz, 2H), 3.75-3.71 (m, 2H), 3.64-3.52 (m, 9H), 3.31 (s, 1H), 3.17-3.13 (m, 3H), 2.98-2.97 (m, 1H), 2.76-2.73 (m, 1H), 2.65-2.62 (m, 1H), 2.20-2.11 (m, 3H); LC-MS: m/z=424 (M+H).sup.+.

[0363] The following compounds were synthesized according to the protocol described above:

TABLE-US-00013 Name Structure NMR, MS # 4-(4-(isoindolin-2-yl)-7- (pyridin-3-yl)-6,7-dihydro- 5H-pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00234] .sup.1H NMR (400 MHz, Chloroform-d) δ 9.03 (d, J = 2.7 Hz, 1H), 8.21 (dd, J = 4.6, 1.4 Hz, 1H), 8.14- 8.09 (m, 1H), 7.31 (s, 4H), 7.26-7.22 (m, 1H), 5.02 (s, 4H), 3.98 (t, J = 8.5 Hz, 2H), 3.79 (s, 8H), 3.47 (t, J = 8.4 Hz, 2H). LCMS (ESI) m/z: 401.1 [M + H].sup.+. 85 4-(4-(4-methylpiperazin-1-yl)-7- (pyridin-3-yl)-6,7-dihydro- 5H-pyrrolo[2,3-d]pyrimidin-2- yl)morpholine [00235] .sup.1H NMR (400 MHz, CD3OD) δ 9.14 (d, J = 2.4 Hz, 1H), 8.14-8.11 (m, 2H), 7.39 (dd, J = 8.4, 4.8 Hz, 1H), 3.98 (t, J = 8.4 Hz, 2H), 3.76-3.70 (m, 12H), 3.22 (t, J = 8.4 Hz, 2H), 2.53 (t, J = 5.0 Hz, 4H), 2.35 (s, 3H). LCMS (ESI) m/z: 382.3 [M + H].sup.+. 60

Synthesis of tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate (Compound 105), 4-(7-phenyl-4-(piperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 88) and 4-(4-(1-methylpiperidin-4-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 65)

[0364] ##STR00236##

Step 1: Synthesis of tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate

[0365] A mixture of 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 0.63 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (391 mg, 1.26 mmol), Cs.sub.2CO.sub.3 (619 mg, 1.90 mmol), Pd.sub.2(dba).sub.3 (58 mg, 0.063 mmol) and P(Cy).sub.3 (35 mg, 0.126 mmol) in CH.sub.3CN (20 mL)/H.sub.2O (5 mL) was stirred at 100° C. for 4 h under nitrogen atmosphere. The mixture was concentrated and the residue was extracted with EtOAc (20*3 mL)/H2O (10 mL). The organic layer was washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by SGC (PE/EA=4:1) to obtain tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (160 mg, 55%) as yellow solid. LCMS (ESI) m/z: 464.3 [M+H].sup.+.

Step 2: Synthesis of tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate

[0366] To a solution of tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (160 mg, 0.35 mmol) in MeOH (20 mL) was added 10% Pd/C (16 mg) and the resultant mixture was stirred at room temperature for 1 h under hydrogen atmosphere. The mixture was filtered and concentrated and crude product obtained was purified by prep-HPLC (0.05% NH4HCO3/H2O:CH3CN=5%˜95%) to obtain tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate (130 mg, 81%) as yellow solid. .sup.1H NMR (400 MHz, CDCl3) δ 7.78 (d, J=8.0 Hz, 2H), 7.39 (t, J=8.0 Hz, 2H), 7.05 (t, J=7.2 Hz, 1H), 4.22 (bs, 2H), 4.06 (t, J=8.4 Hz, 2H), 3.79 (s, 8H), 3.06-3.02 (m, 2H), 2.86-2.79 (m, 2H), 2.61-2.60 (m, 1H), 1.89-1.82 (m, 2H), 1.73-1.69 (m, 2H), 1.51 (s, 9H); LCMS (ESI) m/z: 466.2 [M+H].sup.+.

Step 3: Synthesis of 4-(7-phenyl-4-(piperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0367] To a solution of tert-butyl 4-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-1-carboxylate (100 mg, 0.2 mmol) in dichloromethane (2 mL) was added TFA (0.5 mL) at 0° C. The mixture was then stirred at room temperature for 2 h and concentrated. The residue was purified by prep-HPLC (0.05% NH4HCO3/H2O:CH3CN=5%˜95%) to obtain 4-(7-phenyl-4-(piperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (55 mg, 70%,) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 7.79 (d, J=8.0 Hz, 2H), 7.38 (t, J=8.0 Hz, 2H), 7.05 (t, J=7.2 Hz, 1H), 4.06 (t, J=8.0 Hz, 2H), 3.80 (s, 8H), 3.27 (d, J=12.0 Hz, 2H), 3.04 (t, J=8.4 Hz, 2H), 2.82-2.76 (m, 2H), 2.64-2.60 (m, 1H), 1.97-1.89 (m, 2H), 1.69-1.66 (m, 2H); LCMS (ESI) m/z: 366.1 [M+H].sup.+.

Step 4: Synthesis of 4-(4-(1-methylpiperidin-4-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0368] To a solution of 4-(7-phenyl-4-(piperidin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (40 mg, 0.11 mmol) in methanol (5 mL) was added formaldehyde (4 mg, 0.12 mmol). The mixture was stirred at room temperature for 2 h followed by the addition of sodium cyanoborohydride (35 mg, 0.55 mmol) to the mixture. The mixture was stirred further for 12 h at room temperature and concentrated. The resultant crude product was purified by prep-HPLC (0.05% NH4HCO3/H2O:CH3CN=5%˜95%) to obtain 4-(4-(1-methylpiperidin-4-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (35.9 mg, 85%) as white solid. .sup.1H NMR (400 MHz, CDCl3) δ 7.78 (d, J=8.0 Hz, 2H), 7.38 (t, J=8.0 Hz, 2H), 7.04 (t, J=7.2 Hz, 1H), 4.05 (t, J=8.4 Hz, 2H), 3.79 (s, 8H), 3.06-2.99 (m, 4H), 2.43 (bs, 1H), 2.34 (s, 3H), 2.09-2.00 (m, 4H), 1.74-1.63 (m, 2H); LCMS (ESI) m/z: 380.3 [M+H].sup.+.

Synthesis of 4,4′-(7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-2,4-diyl)dimorpholine (Compound 66)

[0369] ##STR00237##

[0370] To a solution of morpholine (45 mg, 0.52 mmol) in THE (10 mL) was added NaH (38 mg, 0.95 mmol) at 0° C. The suspension was stirred at room temperature for 15 min followed by the addition 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (150 mg, 0.47 mmol) to the mixture. Then mixture was then stirred at 80° C. for 16 h and quenched with water (10 mL), extracted with ethyl acetate (30*3 mL), washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by prep-HPLC (0.05% NH4HCO3/H2O:CH3CN=5%˜95%) to obtain 4,4′-(7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine-2,4-diyl)dimorpholine (18.6 mg, 11%) as yellow solid. .sup.1H NMR (400 MHz, CDCl3) δ 7.72 (d, J=8.0 Hz, 2H), 7.36 (t, J=8.0 Hz, 2H), 7.01 (t, J=7.6 Hz, 1H), 3.98 (t, J=8.4 Hz, 1H), 3.87-3.74 (m, 12H), 3.64-3.62 (m, 4H), 3.16 (t, J=8.4 Hz, 2H); LCMS (ESI) m/z: 368.1 [M+H].sup.+.

Synthesis of 4-(6-(1-methylpyrrolidin-3-yl)-9-phenyl-9H-purin-2-yl)morpholine (Compound 67)

[0371] ##STR00238##

Step 1: Synthesis of 2,6-dichloro-9-phenyl-9H-purine

[0372] To a solution of 2,6-dichloro-9H-purine (1.88 g, 10 mmol), phenylboronic acid (1.83 g, 15 mmol) in dichloromethane (50 mL) were added cupric acetate (900 mg, 5 mmol) and 1,10-Phenanthroline (900 mg, 5 mmol) and the resultant mixture was stirred at room temperature for 2 d under oxygen. The mixture was filtered and the filtrate was concentrated. The residue was subjected to flash chromatography eluting with 0-5% methanol in dichloromethane to obtain 2,6-dichloro-9-phenyl-9H-purine as white solid (1.1 g, 42%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.40 (s, 1H), 7.60-7.60 (m, 2H), 7.56-7.46 (m, 2H), 7.48-7.44 (m, 1H); LCMS (ESI) m/z: 265.0 [M+H].sup.+.

Step 2: Synthesis of tert-butyl 4-(2-chloro-9-phenyl-9H-purin-6-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate

[0373] To a solution of 2,6-dichloro-9-phenyl-9H-purine (132 mg, 0.5 mmol) in dioxane (5 mL) and water (1 mL) were added tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate (148 mg, 0.5 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (40 mg, 0.05 mmol) and sodium carbonate (159 mg, 1.5 mmol) at 25° C. and the resultant mixture was stirred at 80° C. for 6 h under argon protection. It was cooled and the mixture was diluted with water (20 mL). The resultant precipitate was collected by filtration, washed with water (20 mL) and dried to give tert-butyl 4-(2-chloro-9-phenyl-9H-purin-6-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate as yellow solid. (180 mg, 90%). LCMS (ESI) m/z: 342.1 [M−56+H].sup.+.

Step 3: Synthesis of tert-butyl 4-(2-morpholino-9-phenyl-9H-purin-6-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate

[0374] To a mixture of tert-butyl 4-(2-chloro-9-phenyl-9H-purin-6-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate (40 mg, 0.1 mmol) in N,N-dimethylacetamide (2 mL) was added morpholine (44 mg, 0.5 mmol) and the mixture was stirred at 100° C. for 16 h. It was then extracted with ethyl acetate (10 mL*3) and washed with water (10 mL*3). The combined organic layer was dried and concentrated. The residue was subjected to prep-TLC (UV254, Silica, petroleum ether/ethyl acetate=1/1) to give tert-butyl 4-(2-morpholino-9-phenyl-9H-purin-6-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate as yellow solid. (20 mg, 44%). LCMS (ESI) m/z: 449.1 [M+H].sup.+.

Step 4: Synthesis of tert-butyl 3-(2-morpholino-9-phenyl-9H-purin-6-yl)pyrrolidine-1-carboxylate

[0375] To a mixture of tert-butyl 4-(2-morpholino-9-phenyl-9H-purin-6-yl)-2,3-dihydro-1H-pyrrole-1-carboxylate (45 mg, 0.1 mmol) in methanol (5 mL) was added palladium/carbon (10%, 20 mg) and the suspension was stirred at room temperature for 2 h under hydrogen. The mixture was filtered and the filtrate was concentrated to obtain tert-butyl 3-(2-morpholino-9-phenyl-9H-purin-6-yl)pyrrolidine-1-carboxylate as yellow solid. (45 mg, 99%). LCMS (ESI) m/z: 451.2 [M+H].sup.+.

Step 5: Synthesis of 4-(9-phenyl-6-(pyrrolidin-3-yl)-9H-purin-2-yl)morpholine

[0376] A mixture of tert-butyl 3-(2-morpholino-9-phenyl-9H-purin-6-yl)pyrrolidine-1-carboxylate (45 mg, 0.1 mmol) and hydrochloric acid/dioxane (4M, 2 mL) in dichloromethane (5 mL) was stirred at room temperature for 2 h. It was then diluted with 10 mL of dichloromethane and the mixture was washed with aqueous sodium bicarbonate solution (10 mL). The organic layer was concentrated to obtain 4-(9-phenyl-6-(pyrrolidin-3-yl)-9H-purin-2-yl)morpholine as yellow solid. (35 mg, 99%). LCMS (ESI) m/z: 351.2 [M+H].sup.+.

Step 6: Synthesis of 4-(6-(1-methylpyrrolidin-3-yl)-9-phenyl-9H-purin-2-yl)morpholine

[0377] To a solution of 4-(9-phenyl-6-(pyrrolidin-3-yl)-9H-purin-2-yl)morpholine (35 mg, 0.1 mmol) and formaldehyde (35%, 5 drops) in methanol (1 mL) and dichloroethane (2 mL) was added a drop of acetic acid and the mixture was stirred for 1 h. Then sodium cyanoborohydride (31 mg, 0.5 mmol) was added and the resultant mixture was stirred for 16 h at room temperature. The reaction was quenched with water (10 mL) and the mixture was extracted with dichloromethane (10 mL*2). The organic phase was concentrated and the crude product was purified by prep-HPLC (BOSTON pHlex ODS 10 um 21.2×250 mm120 A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to afford 4-(6-(1-methylpyrrolidin-3-yl)-9-phenyl-9H-purin-2-yl)morpholine (6.5 mg, 18%) as white solid. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 8.40 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.60 (t, J=8.0 Hz, 2H), 7.47 (t, J=7.6 Hz, 1H), 4.19 (pent, J=8.4 Hz, 1H), 3.88-3.77 (m, 8H), 3.26 (t. J=9.2 Hz, 1H), 3.08-2.83 (m, 3H), 2.52 (s, 3H), 2.44-2.37 (m, 2H); LCMS (ESI) m/z: 365.3 [M+H].sup.+.

[0378] The following compound was synthesized using similar protocols described above:

TABLE-US-00014 Name Structure NMR, MS # 4-(6-(1- methylpiperidin- 3-yl)-9- (pyridin-3-yl)-9H- purin-2-yl) morpholine [00239] .sup.1H NMR (400 MHz, CD.sub.3OD) δ 9.15 (d, J = 2.0 Hz, 1H), 8.65 (d, J = 4.8 Hz, 1H), 8.43 (s, 1H), 8.38 (d, J = 8.8 Hz, 1H), 7.69 (dd, J = 8.4, 5.2 Hz, 1H), 4.37 (bs, 4H), 3.85-3.83 (m, 4H), 3.23-2.94 (m, 3H), 2.43-2.38 (m, 4H), 2.16-1.63 (m, 5H); LCMS (ESI) m/z: 380.3 [M + H]+. 68

Synthesis of 4-(9-phenyl-6-(pyridin-4-yl)-9H-purin-2-yl)morpholine (Compound 69)

[0379] ##STR00240##

Step 1: Synthesis of 2-chloro-9-phenyl-6-(pyridin-4-yl)-9H-purine

[0380] To a solution of 2,6-dichloro-9-phenyl-9H-purine (264 mg, 1 mmol) in dioxane (10 mL) and water (2 mL) were added pyridin-4-ylboronic acid (123 mg, 1 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (81 mg, 0.1 mmol) and potassium carbonate (414 mg, 3 mmol) at 25° C. and the resultant mixture was stirred at 90° C. for 16 h under argon protection. The mixture was extracted with ethyl acetate (20 mL*3) and washed with water (20 mL). The organic layer was concentrated and the crude product was purified by prep-TLC (Silica, UV254, ethyl acetate/petroleum ether=311) to afford 2-chloro-9-phenyl-6-(pyridin-4-yl)-9H-purine as yellow solid. (50 mg, 16%). LCMS (ESI) m/z: 308.1 [M+H].sup.+. (This step also produced 9-phenyl-2,6-di(pyridin-4-yl)-9H-purine (13 mg, 4%) as the biproduct).

Step 2: Synthesis of 4-(9-phenyl-6-(pyridin-4-yl)-9H-purin-2-yl)morpholine

[0381] To a mixture of 2-chloro-9-phenyl-6-(pyridin-4-yl)-9H-purine (31 mg, 0.1 mmol) in N,N-dimethylacetamide (2 mL) was added morpholine (44 mg, 0.5 mmol) and stirred at 100° C. for 16 h. The mixture was purified with Prep-HPLC (BOSTON pHlex ODS 10 um 21.2×250 mm120 A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to give 4-(9-phenyl-6-(pyridin-4-yl)-9H-purin-2-yl)morpholine as a yellow solid. (26 mg, 72% yield). .sup.1H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J=6.0 Hz, 2H), 8.79 (s, 1H), 8.67 (d, J=6.0 Hz, 2H), 7.94 (d, J=7.6 Hz, 2H), 7.63 (t, J=8.0 Hz, 2H), 7.49 (t, J=7.6 Hz, 1H), 3.83-3.72 (m, 8H); LCMS (ESI) m/z: 359.2 [M+H].sup.+.

Synthesis of 4-(7-phenyl-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-2-yl)morpholine (Compound 70)

[0382] ##STR00241##

Step 1: Synthesis of 2-chloro-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidine

[0383] To a solution of 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (15 g, 77 mmol) in dioxane/water (200 mL/40 mL) were added pyridin-4-ylboronic acid (5.8 g, 77 mmol), potassium carbonate (21.3 g, 154 mmol) and [1,1′bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (5.6 g, 7.7 mmol) at 25° C. and the resultant mixture was stirred at 85° C. for 2 h under argon protection. The mixture was then filtered and the filtrate was concentrated to obtain the target product as dark solid (15 g, 84%). LCMS (ESI) m/z: 231.1 [M+H].sup.+.

Step 2: Synthesis of 7-bromo-2-chloro-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidine

[0384] A mixture of 2-chloro-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidine (3.0 g, 13 mmol) and N-bromosuccinimide (2.3 g, 13 mmol) in N,N-dimethylformamide (30 mL) was stirred at 25° C. for 2 h. To the mixture was added methanol (100 mL) and it was filtered and the filtrate concentrated. The resultant residue was subjected to silica gel column chromatography (petroleum ether:ethyl acetate=1:2) to obtain the target product as yellow solid (2 g, 50%). LCMS (ESI) m/z: 309.2 [M+H].sup.+.

Step 3: Synthesis of 4-(7-bromo-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-2-yl)morpholine

[0385] A mixture of 7-bromo-2-chloro-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidine (0.3 g, 0.97 mmol) and morpholine (0.5 g, 5.8 mmol) in NMP (3 mL) was stirred at 110° C. for 5 h. It was cooled to room temperature and quenched with water (15 mL). The mixture was extracted with ethyl acetate (15 mL*3) and the organic layer was concentrated and subjected to prep-TLC (dichloromethane:acetic ester=1:1) to obtain the target product as yellow solid (0.08 g, 23%). LCMS (ESI) m/z: 360.1 [M+H].sup.+.

Step 4: Synthesis of 4-(7-phenyl-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-2-yl)morpholine

[0386] A mixture of 4-(7-bromo-4-(pyridin-4-yl)-5H-pyrrolo[3,2-d]pyrimidin-2-yl)morpholine (0.07 g, 0.19 mmol), [1,1′bis(diphenylph-osphino)ferrocene]dichloropalladium(II) (0.015 g, 0.02 mmol), cesium carbonate (0.19 g, 0.58 mmol) and phenylboronic acid (0.05 g, 0.39 mmol) in dioxane/water (3 mL/0.5 mL) was stirred at 90° C. for 2 h. The mixture was concentrated and the obtained residue was subjected to prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The mobile phase was acetonitrile/10 mM formic acid aqueous solution) to obtain the target product as yellow solid (0.0198 g, 29%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 11.96 (s, 1H), 8.81 (d, J=5.3 Hz, 2H), 8.38 (bs, 1H), 8.32 (s, 1H), 8.26 (d, J=7.8 Hz, 2H), 8.05 (d, J=5.1 Hz, 2H), 7.42 (t, J=7.6 Hz, 2H), 7.20 (t, J=7.3 Hz, 1H), 3.83-3.74 (m, 8H); LCMS (ESI) m/z: 358.2 [M+H].sup.+.

Synthesis of 2-methyl-1-(4-((1-methylpiperidin-3-yl)methoxy)-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)propan-2-ol (Compound 79)

[0387] ##STR00242##

Step 1: Synthesis of 1-(6-chloro-5-(2-chloroethyl)-2-morpholinopyrimidin-4-ylamino)-2-methylpropan-2-ol

[0388] To a stirred solution of 4-(4,6-dichloro-5-(2-chloroethyl)pyrimidin-2-yl)morpholine (200 mg, 0.674 mmol) and 1-amino-2-methylpropan-2-ol (60 mg, 0.674 mmol) in acetonitrile (20 mL) was added N-ethyl-N-isopropylpropan-2-amine (218 mg, 1.687 mmol) at room temperature. The reaction mixture was then refluxed for 48 h. After cooling to room temperature, the mixture was diluted with ethyl acetate (100 mL), washed with water (30 mL) and brine (30 mL). The organics were dried over sodium sulfate, filtered and concentrated to give 1-(6-chloro-5-(2-chloroethyl)-2-morpholinopyrimidin-4-ylamino)-2-methylpropan-2-ol (200 mg, 85%) as brown solid. LCMS (ESI) m/z: 348.9 [M+H].sup.+.

Step 2: Synthesis of 1-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)-2-methylpropan-2-ol

[0389] Cesium carbonate (466 mg, 1.43 mmol) was added to a solution of 1-(6-chloro-5-(2-chloroethyl)-2-morpholinopyrimidin-4-ylamino)-2-methylpropan-2-ol (200 mg, 0.573 mmol) and sodium iodide (17 mg, 0.113 mmol) in acetonitrile (20 mL) at room temperature. The reaction mixture was refluxed for 4 h under nitrogen atmosphere, cooled and then diluted with ethyl acetate (150 mL). The mixture was washed with water (50 mL), brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column chromatography, eluting with dichloromethane/methanol=9/1 to give 1-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)-2-methylpropan-2-ol (100 mg, 55%) as white solid. LCMS (ESI) m/z: 313.1 [M+H].sup.+.

Step 3: Synthesis of 2-methyl-1-(4-((1-methylpiperidin-3-yl)methoxy)-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)propan-2-ol

[0390] A suspension of (1-methylpiperidin-3-yl)methanol (83 mg, 0.64 mmol) and sodium hydride (32 mg, 0.8 mmol) in tetrahydrofuran (10 mL) was stirred at room temperature for 10 min and then 1-(4-chloro-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)-2-methylpropan-2-ol (100 mg, 0.32 mmol) was added. The resultant mixture was refluxed for 48 h and cooled. It was then diluted with ethyl acetate (80 mL), washed with water (30 mL) and brine (30 mL), dried over sodium sulfate, filtered and concentrated. The crude product was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 2-methyl-1-(4-((1-methylpiperidin-3-yl)methoxy)-2-morpholino-5H-pyrrolo[2,3-d]pyrimidin-7(6H)-yl)propan-2-ol (14 mg, 11%) as pale yellow solid. .sup.1H NMR (500 MHz, MeOD) δ 6.03 (s, 1H), 4.17-4.07 (m, 2H), 3.73-3.61 (m, 10), 3.22 (s, 2H), 2.93-2.77 (m, 4H), 2.27 (s, 3H), 2.07 (bs, 1H), 1.95-1.89 (m, 1H), 1.76-1.57 (m, 4H), 1.23 (s, 6H), 1.00 (m, 1H). LCMS (ESI) m/z: 406.2 [M+H].sup.+.

Synthesis of cyclopropyl(3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-1-yl)methanone (Compound 86)

[0391] ##STR00243##

Step 1: Synthesis of tert-butyl 3-(((trifluoromethyl)sulfonyl)oxy)-2,5-dihydro-1H-pyrrole-1-carboxylate

[0392] A solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (1400 mg, 7.567 mmol) and N,N-diisopropylethylamine (2928 mg, 22.701 mmol) in dichloromethane (50 mL) was cooled to −78° C. and stirred for 10 mins. Then trifluoromethanesulfonic anhydride (2560 mg, 9.081 mmol) was added and the mixture was warmed up and stirred at 25° C. for 16 h. The reaction was quenched with aqueous ammonium chloride solution and extracted with dichloromethane (50 mL×3). The organic layer was dried and concentrated to give tert-butyl 3-(((trifluoromethyl) sulfonyl)oxy)-2,5-dihydro-1H-pyrrole-1-carboxylate (800 mg, 33%) as yellow oil. LC-MS: m/z=262 (M−56+H).sup.+.

Step 2: Synthesis of tert-Butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate

[0393] A solution of tert-butyl 3-(((trifluoromethyl)sulfonyl)oxy)-2,5-dihydro-1H-pyrrole-1-carboxylate (2800 mg, 8.832 mmol), 4,4,4′,4′, 5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (4487 mg, 17.665 mmol), [1,1′-bis(diphenylphosphino) ferrocene] dichloro palladium(II) (325 mg, 0.441 mmol) and potassium acetate (2600 mg, 26.532 mmol) in dioxane (80 mL) was stirred at 75° C. for 4 h. Then water was added and the resultant mixture was extracted with ethyl acetate (50 mL×3). The organic layer was dried and concentrated. The crude product obtained was purified by silica gel column (petroleum ether:ethyl acetate from 50:1 to 10:1) to give tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (2050 mg, 78%) as yellow solid. LC-MS: m/z=240 (M−56+H).sup.+.

Step 3: Synthesis of tert-Butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate

[0394] A solution of tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (280 mg, 0.95 mmol), 4-(4-chloro-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 0.63 mmol), bis(diphenylphosphino) ferrocene]dichloropalladium(II) (20 mg, 0.03 mmol) and potassium carbonate (260 mg, 1.89 mmol) in dioxane/water (30 mL) was stirred at 85° C. for 4 h. Then water was added and the mixture was extracted with ethyl acetate (50 mL×3). The organic layer was dried, concentrated and the crude product obtained was purified by silica gel column chromatography (petroleum ether:ethyl acetate from 50:1 to 10:1) to obtain tert-butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (200 mg, 71%) as yellow solid. LC-MS: m/z=450 (M+H).sup.+.

Step 4: Synthesis of tert-Butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidine-1-carboxylate

[0395] A suspension of tert-butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,5-dihydro-1H-pyrrole-1-carboxylate (200 mg, 0.445 mmol) and palladium/carbon (100 mg) in methanol (5 mL) was stirred at 25° C. for 16 h. The mixture was filtered and the filtrate was concentrated and dried to obtain tert-butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidine-1-carboxylate (180 mg, 90%) as yellow solid. LC-MS: m/z=452 (M+H).sup.+.

Step 5: Synthesis of 4-(7-Phenyl-4-(pyrrolidin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0396] A solution of tert-butyl 3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidine-1-carboxylate (420 mg, 0.931 mmol) and HCl in dioxane (4 mL) in dichloromethane (6 mL) was stirred at 25° C. for 2 h. The mixture was concentrated to obtain 4-(7-phenyl-4-(pyrrolidin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl) morpholine (280 mg, 85%) as yellow solid. LC-MS: m/z=352 (M+H).sup.+.

Step 6: Synthesis of cyclopropyl(3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-1-yl)methanone

[0397] A solution of 4-(7-phenyl-4-(pyrrolidin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (80 mg, 0.228 mmol) and triethylamine (69 mg, 0.684 mmol) in dichloromethane (5 mL) was stirred at 25° C. for 10 min. Then cyclopropane carbonyl chloride (28 mg, 0.274 mmol) was added and the resultant mixture was stirred at room temperature for 2 h. It was filtered and the filtrate was concentrated. The crude product obtained was purified by Pre-HPLC(Column Xbridge 21.2*250 mm C18, 10 um, mobile phase A: water (10 mmol/L ammonium bicarbonate) B: acetonitrile) to obtain cyclopropyl(3-(2-morpholino-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrrolidin-1-yl)methanone (43.1 mg, 45%) as yellow solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J=8.0 hz, 2H), 7.37 (t, J=7.2 Hz, 2H), 7.01 (t, J=7.2 Hz, 1H), 4.08-4.05 (m, 2H), 3.93-3.84 (m, 2H), 3.76-3.64 (m, 9H), 3.56-3.49 (m, 2H), 3.07-3.00 (m, 2H), 2.22-2.04 (m, 2H), 1.79-1.76 (m, 1H), 0.75-0.73 (m, 4H); LC-MS: m/z=420 (M+H).sup.+.

[0398] The following compounds were synthesized according to the protocol described above:

TABLE-US-00015 Name Structure NMR, MS # 1-(3-(2- morpholino- 7-phenyl- 6,7-dihydro- 5H- pyrrolo[2,3- d]pyrimidin- 4- yl)pyrrolidin- 1- yl)ethanone [00244] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 8 Hz, 2H), 7.00 (t, J = 7.2 Hz, 1H), 4.07-4.03 (m, 2H), 3.75-3.73 (m, 1H), 3.58-3.49 (m, 9H), 3.43-3.39 (m, 2H), 3.32-3.29 (m, 1H), 3.05-3.00 (m, 2H), 2.17-2.11 (m, 2H), 2.082 (s, 3H); LC-MS: m/z = 394.2 (M + H).sup.+. 87 1-(4-(2- morpholino- 7-phenyl- 6,7-dihydro- 5H- pyrrolo[2,3- d]pyrimidin- 4- yl)piperidin- 1-yl)ethan-1- one [00245] .sup.1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 6.0 Hz, 2H), 7.39 (t, J = 6.4 Hz, 2H), 7.05 (t, J = 6.0 Hz, 1H), 4.74 (d, J = 10.8 Hz, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.93 (d, J = 10.4 Hz, 1H), 3.76 (s, 8H), 3.19-3.13 (m, 1H), 3.02 (t, J = 6.8 Hz, 2H), 2.70-2.65 (m, 2H), 2.14 (s, 3H), 1.92-1.73 (m, 4H); LCMS (ESI) m/z: 408.1 [M + H]+. 89

Synthesis of 4-(4-(furan-3-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 109) and 4-(7-(pyridin-3-yl)-4-(tetrahydrofuran-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 90)

[0399] ##STR00246##

Step 1: Synthesis of 4-(4-(furan-3-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0400] To a stirred mixture of 4-(4-chloro-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 0.62 mmol), furan-3-ylboronic acid (211 mg, 1.88 mmol), [1,1′-bis(diphenylphosphino) ferrocene]dichloropalladium(II) (69 mg, 0.094 mmol) in acetonitrile (6 mL) and water (1.5 mL) at 20° C. was added cesium carbonate (615 mg, 1.88 mmol). The resultant mixture was stirred at 80° C. for 18 h under nitrogen and cooled. The reaction mixture was then quenched with water (50 mL) and extracted with dichloromethane (50 mL×2). The combined organic fractions were washed with brine (50 mL), dried over sodium sulfate, filtered and concentrated. The residue was subjected to prep-HPLC to obtain the title compound (28.1 mg, 13%) as white solid. 1H NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.29 (d, J=3.5 Hz, 1H), 8.21 (d, J=8.7 Hz, 1H), 7.95 (s, 1H), 7.51 (t, J=1.7 Hz, 1H), 7.34 (dd, J=8.5, 4.7 Hz, 1H), 6.95 (s, 1H), 4.14 (t, J=8 Hz, 2H), 3.84-3.80 (m, 98), 3.24 (t, J=8 Hz, 2H); LCMS (ESI) m/z: 350.1 [M+H].sup.+.

Step 2: Preparation of 4-(7-(pyridin-3-yl)-4-(tetrahydrofuran-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0401] To a solution of 4-(4-(furan-3-yl)-7-(pyridin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (74 mg, 0.21 mmol) in methanol (25 mL) and acetic ester (25 mL) was added palladium on activated carbon 10% Pd (74 mg). The resultant suspension was stirred at 50° C. for 5 h under hydrogen atmosphere. The reaction mixture was filtered and the filtrate was concentrated. The residue was subjected to prep-HPLC (BOSTON pHlex ODS 10 um 21.2×250 mm 120 A. The mobile phase was acetonitrile/0.1% Ammonium bicarbonate) to obtain 4-(7-(pyridin-3-yl)-4-(tetrahydrofuran-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (16.1 mg, 21%) as white solid. .sup.1H NMR (500 MHz, Chloroform-d) δ 9.08 (d, J=2.8 Hz, 1H), 8.29 (dd, J=4.0, 0.4 Hz, 1H), 8.15 (dt, J=9.2, 0.8 Hz, 1H), 7.30 (dd, J=8.5, 4.7 Hz, 1H), 4.11 (t, J=8.0 Hz, 1H), 4.08-4.00 (m, 3H), 3.96-3.88 (m, 2H), 3.80-3.75 (m, 8H), 3.36 (pent, J=6.4 Hz, 1H), 3.14-3.02 (m, 2H), 2.34-2.26 (m, 1H), 2.22-2.14 (m, 1H). LCMS (ESI) m/z: 354.2 [M+H].sup.+.

Synthesis of 4-(6-methyl-7-(pyridin-4-yl)-4-(tetrahydrofuran-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 91)

[0402] ##STR00247##

Step 1: Synthesis of methyl 5-methyl-2-oxotetrahydrofuran-3-carboxylate. PGP-10

[0403] A solution of methyldihydrofuran-2(3H)-one (25 g, 250 mmol) in tetrahydrofuran (100 mL) was added dropwise to lithium bis(trimethylsilyl)amide (1.6 M in tetrahydrofuran, 330 mL, 528 mmol) at −78° C. After stirring at −78° C. for 10 min, dimethyl carbonate (23.6 g, 263 mmol) was added to the mixture at −78° C. and the reaction mixture was warmed up and stirred at room temperature for 16 h. It was then poured into a mixture of concentrated hydrochloric acid (80 mL) and ice (800 mL), followed by extraction with ethyl acetate (800 mL×2). The organic layer was washed by brine, dried over sodium sulfate, filtered and concentrated to obtain the title compound (40 g). This product was used in the next step without further purification. LCMS (ESI) m/z: 159.1 [M+H].sup.+.

Step 2: Synthesis of 5-(2-hydroxypropyl)-2-morpholinopyrimidine-4,6-diol

[0404] Methyl 5-methyl-2-oxotetrahydrofuran-3-carboxylate (40 g, 250 mmol) was added to a solution of morpholine-4-carboximidamide hydrochloride (31 g, 192 mmol) and sodium methanolate (104 g, 576 mmol) in methanol (150 mL) at room temperature. The reaction mixture was then refluxed for 16 h and cooled. Water (200 mL) was added to the mixture and stirred for 0.5 h, followed by the addition of acetic acid (30 mL) and the mixture was stirred further for 2 h at room temperature. The precipitated solid was filtered and dried to give the title compound (41 g, 83%) as white solid. LCMS (ESI) m/z: 256.2 [M+H].sup.+.

Step 3: Synthesis of 4-(4,6-dichloro-5-(2-chloropropyl)pyrimidin-2-yl)morpholine

[0405] To a solution of 5-(2-hydroxypropyl)-2-morpholinopyrimidine-4,6-diol (41 g, 81 mmol) and N-ethyl-N-isopropylpropan-2-amine (44 mL) in toluene (400 mL) was added phosphorus oxychloride (64 mL) at room temperature. The resultant mixture was stirred at 110° C. for 16 h and concentrated. The residue was then dissolved in ethyl acetate (1600 mL) and washed with water (300 mL×2), brine (300 mL), and dried over sodium sulfate. Concentration and purification of the resultant residue on silica gel column chromatography (petroleum ether/ethyl acetate=10/1) afforded the title compound (38 g, 76%) as off-white solid. LCMS (ESI) m/z: 312.0 [M+H].sup.+.

Step 4: Synthesis of 4-(4-chloro-6-methyl-7-(pyridin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0406] A mixture of pyridin-4-amine (151 mg, 1.60 mmol) and sodium hydride (161 mg, 4.025 mmol) in tetrahydrofuran (40 mL) was refluxed for 1 h. After cooling the mixture to room temperature, 4-(4,6-dichloro-5-(2-chloropropyl)pyrimidin-2-yl)morpholine (500 mg, 1.61 mmol) was added. The resultant mixture was refluxed for 16 h and then poured onto ice water (80 mL) and extracted with ethyl acetate (120 mL×2). The organic layer was washed with brine (50 mL) and dried over sodium sulfate. It was filtered, concentrated and the residue was subjected to silica gel column chromatography (petroleum ether/ethyl acetate=1/1 to 0/100) to obtain 4-(4-chloro-6-methyl-7-(pyridin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 37%) as brown solid. LCMS (ESI) m/z: 332.2 [M+H].sup.+.

Step 5: Synthesis of 4-(4-(furan-3-yl)-6-methyl-7-(pyridin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0407] To a stirred mixture of 4-(4-chloro-6-methyl-7-(pyridin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 0.603 mmol), furan-3-ylboronic acid (135 mg, 1.207 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II):CH.sub.2Cl.sub.2 (49 mg, 0.06 mmol) in acetonitrile (8 mL) and water (2 mL) was added cesium carbonate (393 mg, 1.206 mmol) at room temperature. The resultant reaction mixture was stirred at 80° C. for 4 h under nitrogen and cooled. The reaction was quenched with water (50 mL) and the mixture was extracted with dichloromethane (100 mL×2). The combined extractions were washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated. The resultant residue was subjected to silica gel column chromatography, eluting with petroleum ether/ethyl acetate=1/1 to obtain 4-(4-(furan-3-yl)-6-methyl-7-(pyridin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 91%) as yellow solid. LCMS (ESI) m/z: 364.0 [M+H].sup.+.

Step 6: Synthesis of 4-(6-methyl-7-(pyridin-4-yl)-4-(tetrahydrofuran-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine

[0408] A suspension of 4-(4-(Furan-3-yl)-6-methyl-7-(pyridin-4-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (200 mg, 0.55 mmol) and palladium on activated charcoal (10%, 100 mg) in methanol (20 mL) and ethyl acetate (10 mL) was stirred at 30° C. for 16 h under hydrogen atmosphere. The mixture was filtered through celite and concentrated. The resultant residue was purified by prep-HPLC (SunFire C18, 4.6*50 mm, 3.5 um column Xbridge C18 3.5 μm 4.6×50 mm column. The elution system used was a gradient of 5%˜95% over 1.5 min at 2 ml/min and the solvent was acetonitrile/0.01% aqueous ammonium bicarbonate.) to obtain 4-(6-methyl-7-(pyridin-4-yl)-4-(tetrahydrofuran-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (17.1 mg, 8%) as white solid. .sup.1H NMR (500 MHz, MeOD) δ 8.42 (d, J=6.0 Hz, 2H), 7.83 (d, J=6.0 Hz, 2H), 4.80 (bs, 1H), 4.01-3.97 (m, 1H), 3.89-3.87 (m, 1H), 3.82-3.62 (m, 10H), 3.32-3.30 (m, 1H), 3.27-3.22 (m, 1H), 2.69-2.63 (m, 1H), 2.19-2.10 (m, 2H), 1.25-1.22 (m, 3H). LCMS (ESI) m/z: 368.1 [M+H].sup.+.

Synthesis of 4-(4-(1-methylpyrrolidin-3-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (Compound 98)

[0409] ##STR00248##

[0410] A mixture of 4-(7-phenyl-4-(pyrrolidin-3-yl)-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (70 mg, 0.112 mmol), formaldehyde (13 mg, 0.398 mmol) and sodium cyanoborohydride (25 mg, 0.398 mmol) in methanol (4 mL) was stirred at 25° C. for 2 h. Then water (10 mL) was added and the mixture was extracted with ethyl acetate (30 mL×3). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude product obtained was purified by silica gel column (dichloromethane:methanol from 50:1 to 10:1) to obtain 4-(4-(1-methylpyrrolidin-3-yl)-7-phenyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-2-yl)morpholine (22.4 mg, 28%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.80 (d, J=8.0 Hz, 2H), 7.36 (t, J=7.6 Hz, 2H), 7.00 (t, J=7.2 Hz, 1H), 4.03 (t, J=8.4 Hz, 2H), 3.66 (s, 8H), 3.31-3.27 (m, 2H), 3.01-2.92 (m, 3H), 2.79-2.78 (m, 1H), 2.48-2.44 (m, 1H), 2.31 (s, 3H), 2.09-2.04 (m, 2H); LC-MS: m/z=366.3 (M+H)+.

Example 2. PIKfyve Inhibitory Activity

[0411] PIKfyve Biochemical Assay. The biochemical PIKFyve inhibition assays were run by Carna Biosciences according to proprietary methodology based on the Promega ADP-Glo™ Kinase assay. A full-length human PIKFYVE [1-2098 (end) amino acids and S696N, L932S, Q995L, T998S, S1033A and Q1183K of the protein having the sequence set forth in NCBI Reference Sequence No. NP_055855.2] was expressed as N-terminal GST-fusion protein (265 kDa) using baculovirus expression system. GST-PIKFYVE was purified by using glutathione sepharose chromatography and used in an ADP-Glo™ Kinase assay (Promega). Reactions were set up by adding the test compound solution, substrate solution, ATP solution and kinase solution, each at 4×final concentrations. Reactions were prepared with assay buffer (50 mM MOPS, 1 mM DTT, pH7.2), mixed, and incubated in black 384 well polystyrene plates for 1 hour at room temperature. ADP-Glo™ reagent was then added for 40 minutes, followed by kinase detection reagent for an additional 40 minutes. The kinase activity was evaluated by detecting relative light units on a luminescence plate reader. Samples were run in duplicate from 10 μM to 3 nM. Data was analyzed by setting the control wells (+PIKfyve, no compound) to 0% inhibition and the readout value of background (no PIKfyve) set to 100% inhibition, then the % inhibition of each test solution calculated. IC50 values were calculated from concentration vs % inhibition curves by fitting to a four-parameter logistic curve.

[0412] NanoBRET™ TE Intracellular Kinase Assay, K-8 (Promega) Cell-Based Assay. Intracellular inhibition of PIKfyve was assayed using Promega's NanoBRET™ TE Intracellular Kinase Assay, K-8 according to manufacturer's instructions. A dilution series of test compounds was added for 2 hours to HEK293 cells transfected for a minimum of 20 hours with PIKFYVE-NanoLuc® Fusion Vector (Promega) containing a full-length PIKfyve according to manufacturer's specifications in a 96-well plate. Kinase activity was detected by addition of a NanoBRET™ tracer reagent, which was a proprietary PIKfyve inhibitor appended to a fluorescent probe (BRET, bioluminescence resonance energy transfer). Test compounds were tested at concentrations of 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003 μM. BRET signals were measured by a GloMax® Discover Multimode Microplate Reader (Promega) using 0.3 sec/well integration time, 450BP donor filter and 600LP acceptor filters. Active test compounds that bound PIKfyve and displaced the tracer reduced BRET signal. IC50 values were then calculated by fitting the data to the normalized BRET ratio.

[0413] The results of the PIKfyve inhibition assays are summarized in the table below.

TABLE-US-00016 hPIKfyve hPIKfyve BRET Compound IC.sub.50 (μM).sup.a IC.sub.50 (μM).sup.a 1 +++ +++ 2 +++ 3 +++ +++ 4 +++ 5 +++ ++ 6 ++ 7 + 8 +++ 9 ++ 10 ++++ ++ 11 ++ 12 ++ 13 ++ 14 ++ 15 + 16 +++ ++ 17 +++ 18 +++ 19 ++ 20 ++ 21 +++ ++ 22 ++++ ++ 23 +++ 24 +++ + 25 ++ ++ 26 ++ 27 +++ ++ 29 + 30 ++ 31 32 ++ ++ 34 ++ 35 ++ 37 +++ 38 ++ 39 ++ 40 +++ 41 ++++ +++ 48 + 49 ++ 51 +++ 52 +++ 54 +++ 60 + 64 +++ 73 +++ 75 +++ 76 ++ 110 ++ 111 ++ + .sup.a++++ stands for < 10 nM, +++ stands for 10-100 nM, ++ stands for 100-1000 nM, + stands for 1-10 μM, and — stands for > 10 μM.

Example 3. Viability Assay to Assess TDP-43 Toxicity in FAB1 TDP-43 and PIKfyve TDP-43 Yeast Cells

[0414] Generation of TDP-43 yeast model expressing human PIKfyve. Human PIKFYVE (“entry clone”) was cloned into pAG416GPDccdB (“destination vector”) according to standard Gateway cloning protocols (Invitrogen, Life Technologies). The resulting pAG416GPD-PIKFYVE plasmids were amplified in E. coli and plasmid identity confirmed by restriction digest and Sanger sequencing. Lithium acetate/polyethylene glycol-based transformation was used to introduce the above PIKFYVE plasmid into a BY4741 yeast strain auxotrophic for the ura3 gene and deleted for two transcription factors that regulate the xenobiotic efflux pumps, a major efflux pump, and FAB1, the yeast ortholog of PIKFYVE (MATa, snq2::KILeu2; pdr3::KIura3; pdr1::NATMX; fab1::G418.sup.R, his3; leu2; ura3; met15; LYS2+) (FIG. 2).

[0415] Transformed yeast were plated on solid agar plates with complete synthetic media lacking uracil (CSM-ura) and containing 2% glucose. Individual colonies harboring the control or PIKFYVE TDP-43 plasmids were recovered. A plasmid containing wild-type TDP-43 under the transcriptional control of the GAL1 promoter and containing the hygromycin-resistance gene as a selectable marker was transformed into the fab1::G418.sup.R pAG416GPD-PIKFYVE yeast strain (FIG. 1). Transformed yeast were plated on CSM-ura containing 2% glucose and 200 μg/mL G418 after overnight recovery in media lacking antibiotic. Multiple independent isolates were further evaluated for cytotoxicity and TDP-43 expression levels.

[0416] Viability Assay. A control yeast strain with the wild-type yeast FAB1 gene and TDP-43 (“FAB1 TDP-43”, carries empty pAG416 plasmid), and the “PIKFYVE TDP-43” yeast strain, were assessed for toxicity using a propidium iodide viability assay. Both yeast strains were transferred from solid CSM-ura/2% glucose agar plates into 3 mL of liquid CSM-ura/2% glucose media for 6-8 hours at 30° C. with aeration. Yeast cultures were then diluted to an optical density at 600 nm wavelength (OD.sub.600) of 0.005 in 3 mL of CSM-ura/2% raffinose and grown overnight at 30° C. with aeration to an OD.sub.600 of 0.3-0.8. Log-phase overnight cultures were diluted to OD.sub.600 of 0.005 in CSM-ura containing either 2% raffinose or galactose and 150 μL dispensed into each well of a flat bottom 96-well plates. Compounds formulated in 100% dimethyl sulfoxide (DMSO) were serially diluted in DMSO and 1.5 μL diluted compound transferred to the 96-well plates using a multichannel pipet. Wells containing DMSO alone were also evaluated as controls for compound effects. Tested concentrations ranged from 15 μM to 0.11 M. Cultures were immediately mixed to ensure compound distribution and covered plates incubated at 30° C. for 24 hours in a stationary, humified incubator.

[0417] Upon the completion of incubation, cultures were assayed for viability using propidium iodide (PI) to stain for dead/dying cells. A working solution of PI was made where, for each plate, 1 μL of 10 mM PI was added to 10 mL of CSM-ura (raffinose or galactose). The final PI solution (50 μL/well) was dispensed into each well of a new round bottom 96-well plate. The overnight 96-well assay plate was then mixed with a multichannel pipet and 50 μL transferred to the PI-containing plate. This plate was then incubated for 30 minutes at 30° C. in the dark. A benchtop flow cytometer (Miltenyi MACSquant) was then used to assess red fluorescence (82 channel), forward scatter, and side scatter (with following settings: gentle mix, high flow rate, fast measurement, 10,000 events). Intensity histograms were then gated for “PI-positive” or “PI-negative” using the raffinose and galactose cultures treated with DMSO as controls. The DMSO controls for raffinose or galactose-containing cultures were used to determine the window of increased cell death and this difference set to 100. All compounds were similarly gated and then compared to this maximal window to establish the percent reduction in PI-positive cells. IC50 values were then calculated for compounds that demonstrated a concentration-dependent enhancement of viability by fitting a logistic regression curve.

[0418] Upon induction of TDP-43 in both strains, there was a marked increase in inviable cells (rightmost population) with both FAB1 TDP-43 and PIKFYVE TDP-43, with a more pronounced effect in PIKFYVE TDP-43 (FIGS. 3 and 4).

[0419] PIKfyve Inhibition Suppresses Toxicity in PIKfyve TDP-43 Model. The biochemical PIKFyve inhibition assays were run by Carna Biosciences according to proprietary methodology based on the Promega ADP-Glo™ Kinase assay. A full-length human PIKFYVE [1-2098 (end) amino acids and S696N, L932S, Q995L,T998S, S1033A and Q1183K of accession number NP_055855.2] was expressed as N-terminal GST-fusion protein (265 kDa) using baculovirus expression system. GST-PIKFYVE was purified by using glutathione sepharose chromatography and used in an ADP-Glo™ Kinase assay (Promega).

[0420] Reactions were set up by adding the test compound solution, substrate solution, ATP solution and kinase solution, each at 4×final concentrations. Reactions were prepared with assay buffer (50 mM MOPS, 1 mM DTT, pH7.2), mixed, and incubated in black 384 well polystyrene plates for 1 hour at room temperature. ADP-Glo™ reagent was then added for 40 minutes, followed by kinase detection reagent for an additional 40 minutes. The kinase activity was evaluated by detecting relative light units on a luminescence plate reader. Samples were run in duplicate from 10 uM to 3 nM. Data was analyzed by setting the control wells (+ PIKfyve, no compound) to 0% inhibition and the readout value of background (no PIKfyve) set to 100% inhibition, then the % inhibition of each test solution calculated. IC50 values were calculated from concentration vs % inhibition curves by fitting to a four-parameter logistic curve.

[0421] Activity of APY0201, a known PIKFYVE inhibitor, in FAB1 TDP-43 (FIG. 5) and PIKFYVE TDP-43 (FIG. 6). There was no increase in viable cells in FAB1 TDP-43 across a range of compound concentrations as evidenced by a lack in reduction of the right most population of propidium iodide-positive cells (only 0.23 μM is shown). In the PIKFYVE TDP-43 model, 0.23 μM reduced the population of propidium iodide-positive dead cells, indicating PIKFYVE inhibition ameliorated TDP-43 toxicity. Concentrations ranging from 0.5 mM to less than 100 nM afforded increased viability.

##STR00249##

[0422] A panel of compounds was tested in a biochemical PIKFYVE assay (ADP-Glo™ with full-length PIKfyve) and IC50's determined (nM) (see the Table below). The same compounds were also tested in both FAB1 and PIKFYVE TDP-43 yeast models. Their activity is reported here as “active” or “inactive.” Compounds with low nanomolar potency in the biochemical assay were active in the PIKFYVE TDP-43 yeast model. Compounds that were less potent or inactive in the biochemical assay were inactive in the PIKFYVE TDP-43 model. Compounds that were inactive in the biochemical or PIKFYVE TDP-43 assays were plotted with the highest concentrations tested in that assay.

TABLE-US-00017 FAB1 TDP-43 PIKfyve TDP-43 Structure PIKfyve IC.sub.50 (nM) (active/inactive) (active/inactive) [00250] 7.5 Inactive Active [00251] 12 Inactive Active [00252] 4.9 Inactive Active [00253] 640 Inactive Inactive [00254] 2007 Inactive Inactive [00255] >10000 Inactive Inactive

[0423] Biochemical and Efficacy Assays. A larger set of PIKfyve inhibitors were evaluated in both a PIKfyve kinase domain binding assay (nanobret) and in the PIKFYVE TDP-43 yeast strain. IC50 values (μM) were plotted. Data points are formatted based on binned potency from the nanobret assay as indicated in the legend (FIG. 7). Below is a table of compounds and their biochemical and PIKFYVE TDP-43 IC50 values plotted in FIG. 7.

TABLE-US-00018 PIKFYVE Biochemistry PIKFYVE TDP-43 Structure (IC50, μM) (IC50, μM) [00256] 0.003 0.450 [00257] 0.001 1.390 [00258] 0.007 1.120 [00259] 2.660 >15 [00260] 0.014 0.230 [00261] 8.020 >15 [00262] 9.200 >15 [00263] 0.295 >15 [00264] 1.090 >15 [00265] 0.640 >15 [00266] 0.005 4.720 [00267] 0.018 0.693 [00268] 0.253 9.105 [00269] 0.018 8.214 [00270] 0.032 1.447 [00271] 1.343 >15 [00272] >10 >15 [00273] >10 >15 [00274] 0.085 4.273 [00275] 0.042 2.685 [00276] >10 >15 [00277] 0.767 >15 [00278] >10 5.754

OTHER EMBODIMENTS

[0424] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

[0425] Other embodiments are in the claims.