CYCLIC DINUCLEOTIDES AS AGONISTS OF STIMULATOR OF INTERFERON GENE DEPENDENT SIGNALLING

Abstract

Disclosed herein are new cyclic dinucleotide compounds and compositions and their application as pharmaceuticals for the treatment of disease. Methods of modulation of immune response to disease, and induce Stimulator of Interferon Genes (STING) dependent type I interferon production and co-regulated genes in a human or animal subject are also provided for the treatment diseases such as cancer, particularly metastatic solid tumors and lymphomas, inflammation, allergic and autoimmune disease, infectious disease, and for use as anti-viral agents and vaccine adjuvants.

Claims

1. A compound of structural Formula I ##STR00075## or a salt, ester, tautomer, or prodrug thereof, wherein: A.sub.1 and A.sub.2 are independently selected from CH and N; R.sub.1a and R.sub.1b are independently selected from H and NH.sub.2; R.sub.2 is selected from OH, F, Cl, N.sub.3, and NH.sub.2; R.sub.3 is selected from OH, F, Cl, N.sub.3, and NH.sub.2; R.sub.4a and R.sub.4b are independently selected from NH.sub.2, OH, NHR.sub.5, and OR.sub.5; and R.sub.5 is independently selected from methyl, ethyl, and propyl; with the provisos that: when A.sub.1 is CH, then R.sub.4a is not NHR.sub.5 or OR.sub.5; when A.sub.2 is CH, then R.sub.4b is not NHR.sub.5 or OR.sub.5; and when A.sub.1 and A.sub.2 are both N, then at least one of R.sub.2 and R.sub.3 is not OH.

2. The compound as recited in claim 1, wherein A.sub.1 is CH and A.sub.2 is N.

3. The compound as recited in claim 1, wherein A.sub.1 is N and A.sub.2 is CH.

4. The compound as recited in claim 1, wherein A.sub.1 is CH and A.sub.2 is CH.

5. The compound as recited in claim 1, wherein A.sub.1 is N; A.sub.2 is N; and at least one of R.sub.2 and R.sub.3 is not OH.

6. The compound as recited in claim 5, wherein R.sub.2 is selected from F and Cl.

7. The compound as recited in claim 1, wherein R.sub.4a and R.sub.4b are independently selected from NH.sub.2 and NHR.sub.5.

8. The compound as recited in claim 7, wherein R.sub.2 is selected from F and Cl.

9. The compound as recited in claim 1 wherein the compound has structural Formula II: ##STR00076## or a salt, ester, tautomer, or prodrug thereof, wherein: A.sub.1 and A.sub.2 are independently selected from CH and N; R.sub.1a and R.sub.1b are independently selected from H and NH.sub.2; R.sub.2 is selected from OH, F, and C.sub.1; R.sub.3 is OH; R.sub.4a and R.sub.4b are independently selected from NH.sub.2 and OH; and when A.sub.1 and A.sub.2 are both N, then R.sub.2 is not OH.

10. The compound as recited in claim 9, wherein A.sub.1 is CH and A.sub.2 is N.

11. The compound as recited in claim 9, wherein A.sub.1 is N and A.sub.2 is CH.

12. The compound as recited in claim 9, wherein A.sub.1 is CH and A.sub.2 is CH.

13. The compound as recited in claim 9, wherein A.sub.1 is N, A.sub.2 is N, and R.sub.2 is not OH.

14. The compound as recited in claim 13, wherein R.sub.2 is selected from F and Cl.

15. The compound as recited in claim 9, wherein R.sub.4a and R.sub.4b are independently selected from NH.sub.2 and NHR.sub.5.

16. The compound as recited in claim 15, wherein R.sub.2 is selected from F and Cl.

17. The compound as recited in claim 1, wherein the structure is selected from ##STR00077## or a salt, ester, tautomer, or prodrug of any of the above structures.

18. The compound as recited in claim 1, wherein the structure is selected from ##STR00078## or a salt, ester, tautomer, or prodrug of any of the above structures.

19. The compound as recited in claim 1, wherein the structure is selected from ##STR00079## or a salt, ester, tautomer, or prodrug of any of the above structures.

20. The compound as recited in claim 1, wherein the structure is selected from ##STR00080## or a salt, ester, tautomer, or prodrug of any of the above structures.

21. The compound as recited in claim 1, wherein the structure is selected from ##STR00081## or a salt, ester, tautomer, or prodrug of any of the above structures.

22. The compound as recited in claim 1, wherein the structure is selected from ##STR00082## or a salt, ester, tautomer, or prodrug of any of the above structures.

23. The compound as recited in claim 1, wherein the structure is selected from ##STR00083## or a salt, ester, tautomer, or prodrug of any of the above structures.

24. A compound as recited in claim 1 for use as a medicament.

25. A compound as recited in claim 1 for use in the treatment of cancer.

26. A compound as recited in claim 1 for use in the manufacture of a medicament for the prevention or treatment of a disease or condition ameliorated by the agonism of STING.

27. A drug delivery vehicle comprising a compound as recited in claim 1 conjugated to a targeting moiety.

28. The drug delivery vehicle of claim 27, wherein said targeting moiety is a peptide.

29. The drug delivery vehicle of claim 27, wherein said targeting moiety is a biotin or biotin analog.

30. The drug delivery vehicle of claim 27, wherein said targeting moiety is a protein.

31. The drug delivery vehicle of claim 27, wherein said targeting moiety is transferrin.

32. The drug delivery vehicle of claim 27, wherein said targeting moiety is an antibody.

33. The drug delivery vehicle of claim 32, wherein said antibody is a monoclonal antibody.

34. The drug delivery vehicle of claim 27, wherein said targeting moiety is a nanoparticle.

35. The drug delivery vehicle of claim 34, wherein said nanoparticle is comprised of one or more alpha-hydroxycarboxylic acids.

36. A drug delivery vehicle comprising a compound as recited in claim 1 contained within a container moiety.

37. The drug delivery vehicle of claim 36, wherein said container moiety is a nanoparticle.

38. The drug delivery vehicle of claim 36, wherein said container moiety is a liposome.

39. The drug delivery vehicle of claim 36, wherein said container moiety is a micelle.

40. The drug delivery vehicle of claim 36, wherein said container moiety is a vesicle.

41. The drug delivery vehicle of any one of claims 36 to 40, wherein said container moiety comprises phospholipids.

42. The drug delivery vehicle of any one of claims 27 to 41, further comprising a selectivity moiety.

43. The drug delivery vehicle of claim 42, wherein said selectivity moiety confers selective delivery of said drug delivery vehicle to a particular region of the body of a subject.

44. The drug delivery vehicle of claim 42, wherein said selectivity moiety confers selective delivery of said drug delivery vehicle to a particular organ of the body of a subject.

45. The drug delivery vehicle of claim 42, wherein said selectivity moiety confers selective delivery of said drug delivery vehicle to a tumor.

46. A pharmaceutical composition comprising a compound as recited in claim 1 together with a pharmaceutically acceptable carrier.

47. A pharmaceutical composition comprising a drug delivery vehicle as recited in claim 25 together with a pharmaceutically acceptable carrier.

48. A pharmaceutical composition comprising a drug delivery vehicle as recited in claim 34 together with a pharmaceutically acceptable carrier.

49. A method of agonism of STING comprising contacting STING with a compound as recited in claim 1.

50. A method of agonism of STING comprising contacting STING with a drug delivery vehicle as recited in claim 27.

51. A method of agonism of STING comprising contacting STING with a drug delivery vehicle as recited in claim 36.

52. A method of treatment of a STING-mediated disease comprising the administration of a therapeutically effective amount of a compound as recited in claim 1 to a patient in need thereof.

53. A method of treatment of a STING-mediated disease comprising the administration of a therapeutically effective amount of a drug delivery vehicle as recited in claim 27 to a patient in need thereof.

54. A method of treatment of a STING-mediated disease comprising the administration of a therapeutically effective amount of a drug delivery vehicle as recited in claim 36 to a patient in need thereof.

55. The method as recited in any one of claims 52, 53, and 54 wherein said disease is cancer.

56. The method as recited in claim 55, wherein said cancer is a chemotherapy-resistant cancer.

57. The method as recited in claim 55, wherein said cancer is a radiotherapy-resistant cancer.

58. The method as recited in claim 55, wherein said cancer is selected from melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial carcinoma, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma, one or more gastrointestinal stromal tumors, gastroesophageal carcinoma, colorectal cancer, pancreatic cancer, kidney cancer, hepatocellular cancer, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, and one or more plasma cell neoplasms.

59. The method as recited in claim 55, wherein said cancer is melanoma.

60. The method as recited in claim 55, wherein said cancer is metastatic.

61. The method as recited in claim 55, wherein said cancer is lymphoma.

62. The method as recited in any one of claims 52, 53, and 54 wherein said disease is an autoimmune disease or disorder.

63. The method as recited in any one of claims 52, 53, and 54 wherein said disease is an immune deficiency or defect.

64. The method as recited in any one of claims 52, 53, and 54 wherein said disease is an allergic disease.

65. The method as recited in claim 64, wherein said allergic disease is selected from allergic rhinitis and asthma.

66. The method as recited in any one of claims 52, 53, and 54 wherein said disease is an infectious disease.

67. The method as recited in claim 66, wherein said infectious disease is caused by a bacterial infection.

68. The method as recited in claim 66, wherein said infectious disease is caused by a viral infection.

69. A method of treatment of a STING-mediated disease comprising a. the method of any one of claims 52, 53, and 54; and b. administration of another therapeutic agent.

70. A method for achieving an effect in a patient comprising the administration of a therapeutically effective amount of a compound as recited in claim 1 to a patient, wherein said effect is chosen from induction of transcription of host defense genes, production of a cytokine, release of chemokines, priming of antigen-specific T-cells.

71. The method as recited in claim 70, wherein said effect is production of type I interferon.

72. The method as recited in claim 71, wherein said type I interferon is interferon alpha.

73. The method as recited in claim 71, wherein said type I interferon is interferon beta.

74. A method of inducing STING-dependent type I interferon production in an individual comprising the administration of a sufficient amount of a compound as recited in claim 1 to a patient.

75. A method for administration of a vaccine to a subject comprising: a. administration of an effective amount of said vaccine; and b. administration of an effective amount of a compound as recited in claim 1.

76. The method as recited in claim 75, wherein the subject experiences fewer side effects from said vaccine than in the absence of administration of said compound as recited in claim 1.

77. The method as recited in claim 75, wherein the efficacy of said vaccine is higher than in the absence of administration of said compound as recited in claim 1.

78. The method as recited in claim 75, wherein said method has a prophylactic effect in said subject.

79. The method as recited in claim 75, wherein said method has a curative effect in said patient.

Description

INTERMEDIATE A

(2R,3R,4R,5R)-2-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0330] ##STR00019##

Step 1

[0331] ##STR00020##

(2R,3R,4S,5R)-2-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol

[0332] A mixture of (2R,3R,4S,5R)-2-(4-chloro-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (7.0 g, 24.5 mmol) and 7 N NH.sub.3 in MeOH (70 mL) was stirred at 110 C. for 16 h in a pressure safe steel vessel. The mixture was cooled to RT and the volatiles were removed under reduced pressure. Ten batches of this reaction were run in parallel. The residues were combined and triturated with MeOH (500 ml) to give the title compound as an off-white solid.

Step 2

[0333] ##STR00021##

N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

[0334] To a solution of the product from the previous step (65.2 g, 245 mmol) in pyridine (1.14 L) at 0 C. was added TMSCl (119.8 g, 1.10 mol, 4.5 eq) dropwise over 30 minutes. The mixture was stirred for further 30 minutes at 0 C., and BzCl (6 g, 34.9 mmol, 1.5 eq) was then added dropwise. The resulting mixture was allowed to stir at RT for 16 h, cooled to 0 C. and then quenched with H.sub.2O (200 mL), followed by 25% aq. NH.sub.4OH (500 mL). The volatiles were removed under reduced pressure; the residue was diluted in H.sub.2O (1.5 L) and extracted with EtOAc (32.0 L). The organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (DCM:MeOH=20:1) to afford the title compound (60.7 g, 0.164 mol, 67% over two steps).

[0335] MS(ES.sup.+) C.sub.18H.sub.19N.sub.4O.sub.5 requires: 371, found: 370.8 [M+H].sup.+; .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 11.15 (s, 1H), 8.61 (s, 1H), 8.07 (d, J=7.4 Hz, 2H), 7.74 (d, J=3.8 Hz, 1H), 7.61-7.67 (m, 1H), 7.52-7.58 (m, 2H), 6.69 (d, J=3.6 Hz, 1H), 6.24 (d, J=6.4 Hz, 1H), 5.38 (d, J=6.4 Hz, 1H), 5.18 (d, J=4.8 Hz, 1H), 5.08 (t, J=5.5 Hz, 1H), 4.43 (q, J=6.1 Hz, 1H), 4.09-4.15 (m, 1H), 3.93 (q, J=3.6 Hz, 1H), 3.52-3.68 (m, 2H).

Step 3

[0336] ##STR00022##

N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide

[0337] To a solution of the product from the previous step (60.0 g, 162.0 mmol) in pyridine (420 mL) was added DMTrCl (65.87 g, 194.4 mmol, 1.2 eq). The mixture was stirred at RT for 16 h, diluted with CH.sub.2Cl.sub.2 (1.0 L), washed with sat NaHCO.sub.3 (2500 mL), H.sub.2O (500 mL) and brine (500 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (5/1 petroleum ether/EtOAc to 100% EtOAc) to afford the title compound (89.3 g, 132.8 mmol, 82%) as a white foam.

Step 4

[0338] ##STR00023##

N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-hydroxy-tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (A-4a) and N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-3-hydroxy-4-(tert-butyldimethylsilyloxy)-tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (A-4b)

[0339] To a mixture of the product from the previous step (55 g, 81.76 mmol) and AgNO.sub.3 (22.92 g, 134.9 mmol, 22.7 mL, 1.65 eq) in THF (400 mL) was added TBSCl (78.75 g, 522.48 mmol, 1.76 eq). The reaction mixture was stirred at RT for 5 h, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (Petroleum Ether/EtOAc=10/1 to 2/1) to afford A-4a (70 g, 88.9 mmol, 54.4%) and A-4b (7 g, 8.89 mmol, 5.4%).

[0340] A-4a: .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 11.16 (br s, 1H), 8.58 (s, 1H), 8.07 (d, J=7.7 Hz, 2H), 7.52-7.67 (m, 4H), 7.42 (d, J=7.7 Hz, 2H), 7.22-7.32 (m, 7H), 6.88 (d, J=8.8 Hz, 4H), 6.69 (d, J=3.8 Hz, 1H), 6.30 (d, J=5.4 Hz, 1H), 5.12 (d, J=5.8 Hz, 1H), 4.59 (t, J=5.3 Hz, 1H), 4.07-4.21 (m, 2H), 3.73 (s, 6H), 3.28 (br s, 2H), 0.75 (s, 9H), 0.04 (s, 3H), 0.16 (s, 3H).

[0341] A-4b: .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 11.76 (brs, 1H), 8.59 (s, 1H), 8.07 (d, J=7.8 Hz, 2H), 7.61-7.66 (m, 2H), 7.52-7.57 (m, 2H), 7.38 (br d, J=7.8 Hz, 2H), 7.19-7.32 (m, 7H), 6.86 (d, J=8.7 Hz, 4H), 6.68 (d, J=3.6 Hz, 1H), 6.21 (d, J=5.6 Hz, 1H), 5.38 (br d, J=5.9 Hz, 1H), 4.57 (br d, J=5.1 Hz, 1H), 4.34 (t, J=4.4 Hz, 1H), 4.00 (br d, J=4.1 Hz, 1H), 3.72 (s, 6H), 3.30-3.39 (m, 1H), 3.12-3.16 (m, 1H), 3.14 (br dd, J=4.7, 10.4 Hz, 1H), 0.84 (s, 9H), 0.08 (s, 3H), 0.03 (s, 3H).

Step 5

[0342] ##STR00024##

(2R,3R,4R,5R)-2-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0343] To a solution of A-4b (10.0 g, 12.71 mmol) in CH.sub.2Cl.sub.2 (100 mL) were added were added 3-((bis(diisopropylamino)phosphanyl)-oxy)propanenitrile (4.21 g, 14 mmol, 1.1 eq) and DCI (2.25 g, 19.07 mmol, 1.5 eq). The mixture was stirred at RT for 5 h, diluted with CH.sub.2Cl.sub.2 (100 mL) and washed with sat NaHCO.sub.3 (3100 mL). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (petroleum ether/EtOAc=10/1 to 3/1; 0.5% TEA) to afford the title compound (10.0 g, 9.72 mmol, 76%) as a white foam.

[0344] .sup.1H-NMR (400 MHz, CD.sub.3CN) ppm 9.23 (br s, 1H), 8.52 (br s, 1H), 8.01 (br d, J=5.8 Hz, 2H), 7.60-7.68 (m, 1H), 7.51-7.58 (m, 2H), 7.40-7.50 (m, 3H), 7.18-7.34 (m, 7H), 6.79-6.92 (m, 5H), 6.34-6.48 (m, 1H), 4.97-4.70 (m, 1H), 4.63-4.48 (m, 1H), 4.13 (br d, J=3.9 Hz, 1H), 3.84-3.69 (m, 7H), 3.62-3.41 (m, 4H), 3.27-3.15 (m, 1H), 2.58 (t, J=6.2 Hz, 1H), 2.41 (t, J=6.2 Hz, 1H), 1.12-1.03 (m, 9H), 0.91-0.84 (m, 12H), 0.13 (d, J=16.2 Hz, 3H), 0.05 (s, 3H); .sup.31P NMR (162 MHz, CD.sub.3CN) ppm 149.92, 149.53.

INTERMEDIATE B

(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate

[0345] ##STR00025##

Step 1

[0346] ##STR00026##

(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl hydrogen phosphonate

[0347] Phosphorous acid (15.63 g, 190.6 mmol, 15 eq) was co-evaporated three times with anhydrous pyridine (5 mL) and then dissolved in anhydrous pyridine (75 mL) upon heating to ca. 45 C. The mixture was allowed to cool to RT. A-4a (10.0 g, 12.7 mmol) was added and the mixture was cooled to 0 C. Pivaloyl chloride (15.32 g, 127.07 mmol, 10.0 eq) was slowly added at 0 C. and the resulting mixture was allowed to warm to RT and stirred for 16 h. The reaction mixture was then quenched by 1 M aq. TEAB (100 mL) and extracted with EtOAc (31000 mL). The combined organic layers were washed with 0.5 M aq. TEAB (100 m L), and brine (1000 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=50/1) to afford the title compound as a white foam (8.0 g, 8.38 mmol, 66%).

Step 2

[0348] ##STR00027##

(2R,3R,4R,5R)-5-(4-benzamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate

[0349] The product from the previous step (40.0 g, 42.01 mmol) and H.sub.2O (4.0 g, 222 mmol, 4.0 mL, 5.3 eq) were added to a solution of Cl.sub.2CHCOOH in CH.sub.2Cl.sub.2 (6% v/v, 400 mL) and the reaction mixture was stirred at RT for 0.5 h, then washed with H.sub.2O (4200 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and filtered. SiO.sub.2 gel (80 g, previously treated with TEA) was combined with the filtrate, and the mixture was concentrated under reduced pressure to obtain free flowing SiO.sub.2 gel powder. The residue was purified by SiO.sub.2 gel column chromatography (CH.sub.2Cl.sub.2/MeOH=50/1 to 30/1) to give the title compound as a white solid (15.0 g, 23.08 mmol, 55%);

[0350] MS(ES.sup.+) C.sub.24H.sub.34N.sub.4O.sub.7PSi requires: 549, found: 549.1 [M+H].sup.+; .sup.1H-NMR (400 MHz, 400 MHz, DMSO-d.sub.6) ppm 11.15 (br s, 1H), 8.49-8.74 (m, 1H), 8.08 (d, J=7.6 Hz, 2H), 7.78 (d, J=3.6 Hz, 1H), 7.60-7.67 (m, 1H), 7.51-7.57 (m, 2H), 6.74 (d, J=3.6 Hz, 1H), 6.28 (d, J=6.2 Hz, 1H), 5.75 (s, 1H), 4.56-4.74 (m, 2H), 4.16 (br s, 1H), 3.61-3.76 (m, 2H), 3.03 (q, J=7.2 Hz, 5H), 1.19 (t, J=7.4 Hz, 7H), 0.69 (s, 9H), 0.09 (s, 3H), 0.27 (s, 3H); .sup.31P NMR (162 MHz, DMSO-d.sub.6) ppm 0.72.

INTERMEDIATE C

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl) tetrahydrofuran-3-yl hydrogen phosphonate

[0351] ##STR00028##

Step 1

[0352] ##STR00029##

N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide

[0353] To a solution of N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-9H-purin-6-yl)-benzamide (35 g, 93.7 mmol) in pyridine (180 mL) was added DMTrCl (38.12 g, 112.5 mmol, 1.2 eq) and the resulting mixture was stirred at RT for 16 h. The mixture was then diluted with CH.sub.2Cl.sub.2 (800 mL), washed with sat NaHCO.sub.3 (2400 mL) and brine (400 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (petroleum ether/EtOAc=10/1 to 1/4) to give the title compound as a white solid (53.0 g, 78.4 mmol, 84%).

[0354] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 11.26 (br s, 1H), 8.74 (s, 1H), 8.62 (s, 1H), 8.05 (d, J=7.4 Hz, 2H), 7.60-7.72 (m, 1H), 7.48-7.58 (m, 2H), 7.32 (d, J=7.2 Hz, 2H), 7.14-7.24 (m, 7H), 6.80 (dd, J=6.2, 8.7 Hz, 4H), 6.43 (d, J=20.0 Hz, 1H), 5.73-5.85 (m, 1H), 5.61 (d, J=4.4 Hz, 1H), 4.76-4.99 (m, 1H), 4.14 (br d, J=5.4 Hz, 1H), 3.64-3.79 (m, 7H), 3.19-3.33 (m, 2H).

Step 2

[0355] ##STR00030##

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate

[0356] Phosphorous acid (18.2 g, 222 mmol, 15 eq) was co-evaporated three times with anhydrous pyridine (15 mL) and then dissolved with heating in anhydrous pyridine (150 mL). The mixture was allowed to cool to RT. The product from the previous step (10 g, 14.8 mmol) was added, and the resulting mixture was cooled to 0 C. Pivaloyl chloride (17.85 g, 148 mmol, 10 eq) was slowly added at 0 C. and the resulting mixture was allowed to warm to RT and stirred for 16 h. The reaction mixture was then quenched with 1 M aq. TEAB (150 mL) and extracted with EtOAc (3900 mL). The combined organic layers were washed with 0.5 M aq. TEAB (900 mL), brine (900 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=50/1 to 20/1; 1% TEA) to give the title compound as a white foam (38 g).

Step 3

[0357] ##STR00031##

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl) tetrahydrofuran-3-yl hydrogen phosphonate

[0358] The product from the previous step (38 g, 45.19 mmol) and H.sub.2O (4.0 g, 222 mmol, 4.0 mL, 5 eq) were added to a solution of Cl.sub.2CHCOOH in CH.sub.2Cl.sub.2 (6% v/v, 380 mL) and the reaction mixture was stirred at RT for 0.5 h. The reaction mixture was filtered to give a red solid, which was washed with CH.sub.2Cl.sub.2 (220 mL) to give the title compound as a white solid (15 g, 30.87 mmol, 68%).

[0359] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 11.24 (br s, 1H), 8.78 (s, 1H), 8.73 (s, 1H), 8.02-8.08 (m, 2H), 7.76 (d, J=1.2 Hz, 0.5H), 7.62-7.68 (m, 1H), 7.53-7.59 (m, 2H), 6.69 (s, 1H), 6.46 (dd, J=3.2, 16.6 Hz, 1H), 6.07 (d, J=1.4 Hz, 0.5H), 5.87-5.91 (m, 1H), 5.73-5.78 (m, 1H), 5.17-5.28 (m, 1H), 4.22-4.28 (m, 1H), 3.64-3.84 (m, 2H).

INTERMEDIATE D

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)(phenyl)-methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0360] ##STR00032##

Step 1

[0361] ##STR00033##

N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide

[0362] To a solution of N-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)-benzamide (100 g, 269.3 mmol) in pyridine (500 mL) at 0 C. were added DMAP (1.64 g, 13.46 mmol, 0.05 eq) and DMTrCl (100.4 g, 296.2 mmol, 1.1 eq). The reaction mixture was stirred at RT for 16 h, then quenched by addition of MeOH (500 mL). The volatiles were removed under reduced pressure and the residue was purified by SiO.sub.2 gel chromatography (1/1 petroleum ether/EtOAc to 100% EtOAc) to give the title compound as a white foam (150 g, 223 mmol, 83%).

Step 2

[0363] ##STR00034##

N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (D-2a) and N-(9-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(tert-butyl-dimethylsilyloxy)-3-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (D-2b)

[0364] To a solution of the product from the previous step (200 g, 296.9 mmol) in THF (800 mL) and pyridine (15 mL) were added AgNO.sub.3 (83.2 g, 489.8 mmol, 82 mL, 1.65 eq) and TBSCl (78.7 g, 522.5 mmol, 1.76 eq), and the mixture was stirred at RT for 16 h. The reaction mixture was filtered, diluted with H.sub.2O (1.0 L) and extracted with EtOAc (31.0 L). The combined organic layers were washed with brine (21.0 L), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (petroleum ether/EtOAc=3/1 to 1/1) to give D-2a (114 g, 145 mmol, 49%) and D-2b (53 g, 68 mmol, 23%).

Step 3

[0365] ##STR00035##

(2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-5-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite

[0366] To a solution of D-2b (19.0 g, 24.1 mmol) in MeCN (200 mL) at 0 C. were added 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (7.99 g, 26.5 mmol, 1.1 eq) and DCI (3.42 g, 28.9 mmol, 1.2 eq), and the resulting mixture was stirred for 3 h at RT under N.sub.2 atmosphere. The volatiles were removed under reduced pressure and the residue was purified by SiO.sub.2 gel chromatography (petroleum ether/EtOAc=4/1 to 1.5/1; 1% TEA) to give the title compound as a white foam (20.5 g, 20.74 mmol, 86%).

[0367] MS(ES.sup.+) C.sub.53H.sub.67N.sub.7O.sub.8PSi requires: 988, found: 987.8 [M+H].sup.+; .sup.1H-NMR (400 MHz, CD.sub.3CN) ppm 8.57 (s, 1H), 8.26 (s, 1H), 7.91 (br d, J=7.6 Hz, 2H), 7.57 (m, 1H), 7.47-7.49 (m, 2H), 7.17-7.21 (m, 9H), 6.73-6.76 (m, 4H), 6.10-6.19 (m, 1H), 5.10-5.14 (m, 1H), 4.64-4.68 (m, 1H), 4.10-4.14 (m, 1H), 3.25-3.75 (m, 7H), 3.40-3.51 (m, 4H), 3.15-3.18 (m, 1H), 2.58 (t, J=6.0 Hz, 1H), 2.41 (t, J=6.0 Hz, 1H), 1.03-1.06 (m, 9H), 0.76-0.86 (m, 12H), 0.00-0.93 (m, 6H); .sup.31P NMR (162 MHz, CD.sub.3CN) ppm 150.32, 149.58.

INTERMEDIATE E

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate triethylammonium salt

[0368] ##STR00036##

[0369] Step 1

##STR00037##

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)-(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-3-yl (2-cyanoethyl) hydrogen phosphonate

[0370] To a solution of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)-oxy)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (25 g, 25.3 mmol) in MeCN (12 mL) was added H.sub.2O (912 mg, 50.6 mmol, 2.0 eq) and pyridinium trifluoroacetate (5.86 g, 30.36 mmol, 1.2 eq) and the resulting mixture was stirred for 5 minutes at RT. tert-Butylamine (1.85 g, 25.3 mmol, 1.0 eq) was added and the resulting mixture was stirred for further 25 minutes at RT and then concentrated under reduced pressure to give the title compound (23.4 g) as a white foam, which was used in the next step without further purification.

Step 2

[0371] ##STR00038##

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate

[0372] The crude product from the previous step (23.4 g, 25.3 mmol) was added to a solution of Cl.sub.2CHCOOH in CH.sub.2Cl.sub.2 (6% v/v, 200 mL). H.sub.2O (2.28 g, 126 mmol, 5.0 eq) was added and the resulting mixture was stirred for 20 minutes at RT. The reaction mixture was quenched by the addition of pyridine (30 mL) at RT, and was then concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=20/1 to 5/1) to give the title compound (15 g, 24.1 mmol).

Step 3

[0373] ##STR00039##

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate triethylammonium salt

[0374] The product from the previous step was dissolved in MeOH (100 mL), TEA resin (50.0 g; prepared from DOWEX 50WX2-H.sup.+ by washing with deionized water until pH=7; then with 1N aq. TEA; then again with deionized water until pH=7; then MeOH) was added and the mixture was stirred at RT for 0.5 h. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure to give the title compound as a TEA salt (yellow solid; 15 g, 23 mmol, 95%).

[0375] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 8.57 (s, 1H), 8.26 (s, 1H), 7.91 (br d, J=7.6 Hz, 2H), 7.57 (m, 1H), 7.47-7.49 (m, 2H), 7.17-7.21 (m, 9H), 6.73-6.76 (m, 4H), 6.10-6.19 (m, 1H), 5.10-5.14 (m, 1H), 4.64-4.68 (m, 1H), 4.10-4.14 (m, 1H), 3.25-3.75 (m, 7H), 3.40-3.51 (m, 4H), 3.15-3.18 (m, 1H), 2.58 (t, J=6.0 Hz, 1H), 2.41 (t, J=6.0 Hz, 1H), 1.03-1.06 (m, 9H), 0.76-0.86 (m, 12H), 0.00-0.93 (m, 6H).

INTERMEDIATE F

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-chloro-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate

[0376] ##STR00040##

Step 1

[0377] ##STR00041##

[0378] A mixture of (2R,3S,4R,5R)-2-(6-amino-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-3-yl trifluoromethanesulfonate (69.0 g, 0.11 mol, 1.0 eq) and LiCl (51.2 g, 1.21 mol, 11.0 eq) in DMF (600 mL) was heated at 50 C. for 1.5 h. The reaction mixture was diluted with DCM (500 mL) and washed with H.sub.2O (300 mL3). The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (PE:EtOAc=1:1) to give the title compound (46.0 g, 89.5 mmol, 81.4% yield) as a yellow solid.

[0379] .sup.1H NMR (400 MHz CDCl.sub.3) ppm 8.30-8.38 (m, 1H), 8.06 (s, 1H), 6.20 (d, J=5.9 Hz, 1H), 5.82-5.95 (m, 2H), 4.96-5.04 (m, 1H), 4.56-4.63 (m, 1H), 4.17 (q, J=3.5 Hz, 1H), 3.96 (dd, J=11.4, 4.3 Hz, 1H), 3.77 (dd, J=11.4, 3.0 Hz, 1H), 0.92 (d, J=13.9 Hz, 18H), 0.14 (d, J=8.8 Hz, 6H), 0.08 (s, 6H).

Step 2.

[0380] ##STR00042##

[0381] To a solution of the product from previous step (46.0 g, 89.4 mmol, 1.0 eq) in Py (460 mL) at 0 C. was added BzCl (25.2 g, 179 mmol, 20.5 mL, 2.0 eq) dropwise under N.sub.2, and the resulting mixture was stirred at 25 C. for 16 hrs. The reaction was diluted with H.sub.2O (200 mL) and extracted with EtOAc (200 mL3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give N-benzoyl-N-(9-((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3-chlorotetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (60.0 g) as yellow oil which was used for the next step without further purification. MS(ES.sup.+) C.sub.36H.sub.48ClN.sub.5O.sub.5Si.sub.2 requires: 721, found: 722 [M+H].sup.+;

Step 3

[0382] ##STR00043##

[0383] To a solution of the product from previous step (60.0 g, 97 mmol, 1.0 eq) in THF (600 mL) was added TBAF (1 M in THF, 290 mL, 3.0 eq). Then the mixture was stirred at 25 C. for 16 hrs. The volatiles were removed under reduced pressure to give N-benzoyl-N-(9-((2R,3R,4R,5R)-3-chloro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (48.0 g) as a yellow oil which was used for the next step without further purification. MS(ES.sup.+) C.sub.24H.sub.20ClN.sub.5O.sub.5 requires: 493, found: 494 [M+H].sup.+;

Step 4

[0384] ##STR00044##

[0385] To a solution of the product from previous step (48.0 g, 97.2 mmol, 1.0 eq) in THF (500 mL) was added NH.sub.3.H.sub.2O (28% in water, 8.0 mL, 7.28 g, 54.0 mmol, 0.5 eq). The resulting mixture was stirred at 25 C. for 1 hr, during which time a solid product was formed. The suspension was filtered and the filter cake was washed with THF (300 mL3). The filtrate was concentrated under reduced pressure to give N-(9-((2R,3R,4R,5R)-3-chloro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (36 g, 92.3 mmol, 95% yield) as a white solid product; MS(ES.sup.+) C.sub.17H.sub.16ClN.sub.5O.sub.4 requires: 389, found: 390 [M+H].sup.+;

Steps 5 to 7

[0386] ##STR00045##

[0387] The transformations depicted in the above scheme were performed according to the procedures previously described for the synthesis of Intermediate C, steps 1 to 3.

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-chloro-2(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (Intermediate F)

[0388] white solid (13.0 g, 27.8 mmol, 30% over three steps); .sup.1H NMR: (400 MHz DMSO-d.sub.6) ppm 11.28 (br s, 1H), 8.78 (br d, J=4.0 Hz, 2H), 8.05 (br d, J=7.4 Hz, 2H), 7.60-7.68 (m, 1H), 7.55 (br t, J=7.5 Hz, 2H), 6.34 (br d, J=5.9 Hz, 1H), 5.27 (br d, J=5.1 Hz, 1H), 5.02 (br s, 1H), 4.30 (br s, 1H), 3.74 (br s, 2H), 3.01-3.11 (m, 2H); .sup.31P NMR: (162 MHz DMSO-d.sub.6) 0.63 ppm.

Example 1a and Example 1b

Cyclic dinucleotides RR-CD-A-7dA and SR-CD-A-7dA dithio-[R.SUB.p., R.SUB.p.]-cyclic-[A(2,5)p-7dA(3,5)p]dithio-[S.SUB.p., R.SUB.p.]-cyclic-[A(2,5)p-7dA (3,5)p]

[0389] ##STR00046##

Step 1

[0390] ##STR00047##

[0391] Compound 1-1

[0392] To a solution of Intermediate B (4.00 g, 6.16 mmol) in CH.sub.3CN (50 mL) was added pyridine-TFA (2.38 g, 12.3 mmol, 2.0 eq) followed by a mixture of Intermediate D (6.70 g, 6.78 mmol, 1.10 eq) and 3A molecular sieves (1.0 g, 24.6 mmol, 4.0 eq) in CH.sub.3CN (50 mL), and the resulting mixture was stirred for 30 minutes at RT. DDTT (1.52 g, 7.39 mmol, 1.20 eq) was then added and the mixture was stirred at RT for further 30 minutes. The volatiles were removed under reduced pressure to afford the crude compound 1-1 (9.04 g), which was used without further purification in the next step.

Step 2

[0393] ##STR00048##

[0394] Compound 1-2

[0395] To a solution of Cl.sub.2CHCOOH in CH.sub.2Cl.sub.2 (6% v/v, 200 mL) was added H.sub.2O (2.0 g, 111 mmol, 2.0 mL, 18.0 eq) and compound 1-1 from the previous step (9.04 g, assume 6.16 mmol). The reaction mixture was stirred at RT for 0.5 h, then quenched with pyridine (120 mL) and concentrated under reduced pressure, to afford crude compound 1-2 (7.18 g), which was used without further purification in the next step.

[0396] MS(ES.sup.+) C.sub.50H.sub.67N.sub.10O.sub.13P.sub.2SSi.sub.2 requires: 1165, found: 1165.3 [M+H].sup.+.

Step 3

[0397] ##STR00049##

[0398] Compound 1-3

[0399] To a solution of compound 1-2 from the previous step (7.18 g, assume 6.16 mmol) in pyridine (50 mL) was added DMOCP (3.98 g, 21.6 mmol, 3.5 eq). The mixture was stirred for 0.5 h at RT. To the mixture was then added Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 1.85 g, 9.24 mmol, 1.5 eq), and the resulting mixture and stirred at RT for further 30 minutes. The reaction mixture was quenched by addition of 3.4% aq. NaHCO.sub.3 (1.0 L), and then extracted with EtOAc (2500 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=30/1 to 15/1) to give compound 1-3 (3.0 g, 2.04 mmol) as a mixture of diastereoisomers which was used as such in the following step.

Step 4

[0400] ##STR00050##

[0401] Compound 1-4

[0402] To a solution of compound 1-3 from the previous step (3.0 g, 2.04 mmol) in MeOH (30 mL) was added NH.sub.4OH (32.8 g, 935 mmol, 458 eq). The mixture was stirred at 50 C. for 12 h in a pressure safe steel vessel, then concentrated under reduced pressure. The residue was purified by prep-HPLC (PHENOMENEX LUNA C18 250*50 10 um; mobile phase: A: H.sub.2O (10 mM NH.sub.4HCO.sub.3); B: MeCN; A %-B %=20%-50%, 20 minutes) to give two products: compound 1-4a (R.sub.pR.sub.p or S.sub.pR.sub.p diastereoisomer; 380 mg, 0.391 mmol;) and compound 1-4b (S.sub.pR.sub.p or R.sub.pR.sub.p diastereoisomer; 350 mg, 0.349 mmol) as a white solids.

Step 5

[0403] ##STR00051##

[0404] Compound 1-5

[0405] To a solution of compound 1-4a (200 mg, 218 umol) in MeOH (5.0 mL) was added NH.sub.4F (80.7 mg, 2.18 mmol, 10.0 eq) and the resulting mixture was stirred at 60 C. for 16 h. The volatiles were removed under reduced pressure and the residue was purified by prep-HPLC [Waters Xbridge 150*25 5 um; mobile phase: A: H.sub.2O (10 mM NH.sub.4HCO.sub.3); B: MeCN; A %-B %=1%-20%, 10.5 minutes] to give compound 1-5a (30 mg, 40 umol) as a white solid.

[0406] Reaction of compound 1-4b in a similar manner afforded compound 1-5b (30 mg, 40 umol) as a white solid.

Step 6

[0407] ##STR00052##

(1R,6R,8R,9R,10R,12R,15R,17R,18R)-17-(6-amino-9H-purin-9-yl)-8-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-9,18-dihydroxy-3,12-dimercapto-2,4,7,11,13,16-hexaoxa-3.SUP.5., 12.SUP.5.-diphosphatricyclo[13.2.1.0.SUP.6,10.]octadecane-3,12-dione (1-5), disodium salt (Example 1a and Example 1b)

[0408] To a solution of compound 1-5a (30.0 mg, 41.5 umol) in H.sub.2O (5.0 mL) was added DOWEX-50WX8 (Na.sup.+ form; 300 mg) and the mixture was stirred at RT for 0.5 h. The reaction was then filtered, and the filtrate was lyophilized to give Example 1a (R.sub.p, R.sub.p or S.sub.p, R.sub.p 28.0 mg, 38.1 umol) as a white solid.

[0409] MS(ES.sup.+) C.sub.21H.sub.26N.sub.9O.sub.10P.sub.2S.sub.2 requires: 690, found: 690.0 [M+H].sup.+; .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 8.52 (s, 1H), 8.26 (s, 1H), 8.19 (s, 1H), 7.50 (d, J=3.6 Hz 1H), 6.85 (d, J=3.6 Hz, 1H), 6.09-6.13 (m, 2H), 5.46 (d, J=8.8 Hz, 1H), 5.28-5.30 (m, 1H) 4.63 (d, J=4 Hz, 1H), 4.03-4.27 (m, 5H), 3.64-3.69 (m, 2H); .sup.31P NMR (162 MHz, DMSO-d.sub.6) ppm 60.19, 56.77; R.sub.t=1.797 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes]

[0410] Reaction of compound 1-5b in a similar manner gave Example 1b (S.sub.p, R.sub.p or R.sub.p, R.sub.p 30.0 mg, 39.6 umol) as a white solid.

[0411] MS(ES.sup.+) C.sub.21H.sub.26N.sub.9O.sub.10P.sub.2S.sub.2 requires: 690, found: 690.0 [M+H].sup.+; .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 8.50 (s, 1H), 8.29 (s, 1H), 8.19 (s, 1H), 7.62 (d, J=3.2 Hz 1H), 6.85 (d, J=4.0 Hz, 1H), 6.09-6.14 (m, 2H), 5.19-5.29 (m, 2H), 4.68 (dd, J=7.6 Hz, 1H), 4.37 (d, J=4.0 Hz, 1H), 4.11-4.20 (m, 3H), 3.95-3.99 (m, 1H), 3.69-3.81 (m, 2H); .sup.31P NMR (162 MHz, CD.sub.3OD) ppm 59.29, 51.96; R.sub.t=2.101 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

Example 2a and Example 2b

Cyclic dinucleotides RR-CD-A-2F-A and SR-CD-A-2F-A; dithio-[R.SUB.p., R.SUB.p.]-cyclic-[A(2,5)p-2F-A(3,5)p]dithio-[S.SUB.p., R.SUB.p.]-cyclic-[A(2,5)p-2F-A (3,5)p]

[0412] ##STR00053##

Step 1

[0413] ##STR00054##

[0414] Compound 2-1

[0415] To a solution of Intermediate C (4.0 g, 9.15 mmol) in CH.sub.2Cl.sub.2 (40 ml) was added TEA (463 mg, 4.58 mmol, 0.50 eq). The mixture was stirred for 5 minutes at RT and the volatiles were removed under reduced pressure. The residue was dissolved in CH.sub.3CN (40.00 mL) and pyridine-TFA (3.53 g, 18.3 mmol, 2.0 eq) was added, followed by a mixture of Intermediate D (9.04 g, 9.15 mmol, 1.0 eq) and 3A molecular sieves (1.48 g, 36.6 mmol, 4.0 eq) in CH.sub.3CN (40 mL), and the resulting mixture was stirred for 30 minutes at RT. DDTT (2.25 g, 10.98 mmol, 1.2 eq) was added and the mixture was stirred at RT for further 30 minutes. The volatiles were removed under reduced pressure to afford the crude compound 2-1 (12.4 g), which was used without further purification in the next step.

Step 2

[0416] ##STR00055##

[0417] Compound 2-2

[0418] To a solution of Cl.sub.2CHCOOH in CH.sub.2Cl.sub.2 (6% v/v, 200 mL) was added compound 2-1 from the previous step (12.4 g, assume 9.15 mmol) and H.sub.2O (2.00 g, 111 mmol, 2.0 mL, 12.1 eq). The reaction mixture was stirred at RT for 0.5 h, then quenched with pyridine (100 mL) and concentrated under reduced pressure to afford compound 2-2 (9.64 g), which was used without further purification in the next step.

Step 3

[0419] ##STR00056##

[0420] Compound 2-3

[0421] To a solution of compound 2-2 from the previous step (9.63 g, assume 9.15 mmol) in pyridine (200 mL) was added DMOCP (5.90 g, 32.0 mmol, 3.5 eq) and the mixture was stirred for 0.5 h at RT. 3H-1,2-Benzodithiol-3-one 1,1-dioxide (2.75 g, 13.7 mmol, 1.5 eq) was then added, and the resulting mixture and stirred at RT for further 30 minutes. The reaction mixture was quenched by addition of 3.4% aq. NaHCO.sub.3 (1.0 L), and then extracted with EtOAc mL (2500 mL). The combined organic layers were washed with brine (300 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=30/1 to 10/1) to give compound 2-3 (3.2 g, 2.1 mmol) as a mixture of diastereoisomers which was used as such in the following step.

Step 4

[0422] ##STR00057##

[0423] Compound 2-4

[0424] To a solution of compound 2-3 from the previous step (2.0 g, 1.87 mmol) in MeOH (10 mL) was added NH.sub.4OH (18.2 g, 519 mmol, 277 eq). The mixture was stirred at 50 C. for 16 h in a pressure safe steel vessel, then concentrated under reduced pressure. The residue was purified by prep-HPLC [PHENOMENEX LUNA C18 250*50 10 um; mobile phase: A: H.sub.2O (10 mM NH.sub.4HCO.sub.3); B: MeCN; A %-B %=10%-40%, 20 minutes] to give two products: compound 2-4a (R.sub.pR.sub.p or S.sub.pR.sub.p diastereoisomer, 180 mg, 0.209 mmol) and compound 2-4b (S.sub.pR.sub.p or R.sub.pR.sub.p diastereoisomer, 200 mg, 0.228 mmol) as white solids.

Step 5

[0425] ##STR00058##

[0426] Compound 2-5

[0427] To a solution of compound 2-4a (100 mg, 119 umol) in MeOH (3.0 mL) was added NH.sub.4F (44.1 mg, 1.19 mmol, 10 eq) and the resulting mixture was stirred at 60 C. for 16 h. The reaction mixture was then allowed to cool to RT and concentrated under reduced pressure. The residue was taken up in H.sub.2O (0.5 mL), cooled to 10 C. and kept stirring for 30 minutes, then filtered and the filter cake was collected to give compound 2-5a (30.0 mg, 41.3 umol) as a white solid.

[0428] MS(ES.sup.+) C.sub.20H.sub.24FN.sub.10O.sub.9P.sub.2S.sub.2 requires: 693, found: 693.2 [M+H].sup.+; .sup.1H-NMR (400 MHz, CD.sub.3OD) ppm 8.98 (s, 1H), 8.22 (s, 2H), 7.82 (s, 1H), 6.45 (d, J=14.4 Hz, 1H), 6.33 (d, J=8.0 Hz, 1H), 5.62 (d, J=53.8 Hz, 1H), 5.32 (m, 1H), 5.07-5.13 (m, 1H), 4.36-4.46 (m, 5H), 4.06 (d, J=11.2 Hz, 1H), 3.86-3.90 (m, 1H).

[0429] Reaction of compound 2-4b in a similar manner afforded compound 2-5b (30.0 mg, 41.3 umol) as a white solid.

[0430] MS(ES.sup.+) C.sub.20H.sub.24FN.sub.10O.sub.9P.sub.2S.sub.2 requires: 693, found: 693.2[M+H].sup.+; .sup.1H-NMR (400 MHz, CD.sub.3OD) ppm 8.76 (s, 1H), 8.49 (s, 1H), 8.24 (s, 1H), 8.18 (s, 1H), 6.34-6.41 (m, 2H), 5.70 (dd, J=51.8 Hz, 1H), 5.22-5.239 (m, 2H), 4.50-4.59 (m, 4H), 4.32 (s, 1H), 4.03-4.07 (m, 1H).

Step 6

[0431] ##STR00059##

(1R,6R,8R,9R,10R,12R,15R,17R,18R)-8,17-di(6-amino-9H-purin-9-yl)-9-fluoro-18-hydroxy-3,12-dimercapto-2,4,7,11,13,16-hexaoxa-3.SUP.5., 12.SUP.5.-diphosphatricyclo-[13.2.1.0.SUP.6,10.]octadecane-3,12-dione (2-5), disodium salt (Example 2a and Example 2b)

[0432] To a solution of compound 2-5a (30.0 mg, 41.3 umol) in H.sub.2O (10.0 mL) was added Dowex-50WX8 (Na.sup.+ form; 300 mg) and the mixture was stirred at RT for 4 h. The reaction was then filtered, and the filtrate was lyophilized to give Example 2a (R.sub.pR.sub.p or S.sub.pR.sub.p; 30.0 mg, 40.6 umol) as a white solid.

[0433] MS(ES.sup.+) C.sub.20H.sub.24FN.sub.10O.sub.9P.sub.2S.sub.2 requires: 693, found: 693.0 [M+H].sup.+; .sup.1H-NMR (400 MHz, DMSO-d.sub.6) ppm 8.56 (s, 1H), 8.40 (s, 1H), 8.17 (s, 1H), 8.13 (s, 1H), 7.34 (s, 2H), 7.22 (s, 2H), 6.23 (m, 1H), 6.09 (d, J=8.4 Hz 1H), 5.71 (d, J=52.8 Hz, 1H), 5.54 (s, 1H), 5.15-5.30 (m, 2H), 3.91-4.37 (m, 5H), 3.67-3.70 (m, 1H); .sup.31P NMR (162 MHz, CD.sub.3OD) ppm 55.97, 53.66; R.sub.t=1.384 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

[0434] Reaction of compound 2-5b in a similar manner gave Example 2b (S.sub.pR.sub.p or R.sub.pR.sub.p, 28.0 mg, 40.6 umol) as a white solid.

[0435] MS(ES.sup.+) C.sub.20H.sub.24FN.sub.10O.sub.9P.sub.2S.sub.2 requires: 693, found: 693.0 [M+H].sup.+; .sup.1H-NMR (400 MHz, CD.sub.3OD) ppm 8.86 (br s, 1H), 8.39 (s, 1H), 8.19 (s, 1H), 8.02 (br s, 1H), 6.35-6.41 (m, 2H), 5.70 (d, J=51.8 Hz, 1H), 5.22-5.27 (m, 2H), 4.35-4.60 (m, 5H), 4.05-4.08 (m, 2H); .sup.31P NMR (162 MHz, CD.sub.3OD) ppm 57.39, 52.28; R.sub.t=1.644 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

Example 3a and Example 3b

Cyclic dinucleotides RR-CD-7dA-A and SR-CD-7dA-A dithio-[R.SUB.p., R.SUB.p.]-cyclic-[7dA(2,5)p-A(3,5)p]dithio-[S.SUB.p., R.SUB.p.]-cyclic-[7dA(2,5)p-A (3,5)p]

[0436] ##STR00060##

Step 1

[0437] ##STR00061##

[0438] Compound 3-1

[0439] To a solution of Intermediate E (3.0 g, 4.61 mmol) in CH.sub.3CN (30 mL) was added pyridine-TFA (1.78 g, 9.22 mmol, 2.0 eq) followed by a mixture of Intermediate A (5.0 g, 5.07 mmol, 1.1 eq) and molecular sieves (0.8 g, 18.4 mmol, 4.00 eq) in CH.sub.3CN (30 mL), and the resulting mixture was stirred for 30 minutes at RT. DDTT (1.14 g, 5.53 mmol, 1.2 eq) was then added and the mixture was stirred at RT for further 30 minutes. The volatiles were removed under reduced pressure to afford crude compound 3-1 (6.77 g), which was used without further purification in the next step.

Step 2

[0440] ##STR00062##

[0441] Compound 3-2

[0442] To a solution of Cl.sub.2CHCOOH acid in CH.sub.2Cl.sub.2 (6% v/v, 100 mL) was added H.sub.2O (83.1 mg, 4.61 mmol, 83.12 uL, 1.00 eq) and compound 3-1 from the previous step (6.77 g, assume 4.61 mmol, 1.00 eq). The reaction mixture was stirred at RT for 0.5 h, then quenched with pyridine (80 mL) and concentrated under reduced pressure to afford compound 3-2 (5.48 g), which was used without further purification in the next step.

Step 3

[0443] ##STR00063##

[0444] Compound 3-3

[0445] To a solution of compound 3-2 from the previous step (5.48 g, assume 4.61 mmols) in pyridine (150 mL) was added DMOCP (4.77 g, 25.8 mmol, 5.5 eq) and the mixture was stirred for 0.5 h at RT. 3H-1,2-Benzodithiol-3-one 1,1-dioxide (1.41 g, 7.05 mmol, 1.5 eq) was then added, and the resulting mixture and stirred at RT for further 30 minutes. The reaction mixture was quenched by addition of 3.4% aq. NaHCO.sub.3 (600 mL), and then extracted with EtOAc (2300 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=30/1 to 15/1) to give compound 3-3 (3.0 g, 1.53 mmol) as a mixture of diastereoisomers which was used as such in the following step.

Step 4

[0446] ##STR00064##

[0447] Compound 3-4

[0448] To a solution of compound 3-3 from the previous step (3.0 g, 1.53 mmol) in MeOH (30 mL) was added NH.sub.4OH (16.4 g, 468 mmol, 307 eq). The mixture was stirred at 50 C. for 16 h in a pressure safe steel vessel, then concentrated under reduced pressure. The residue was purified by prep-HPLC [PHENOMENEX LUNA C18 250*50 10 um; mobile phase: A: H.sub.2O (10 mM NH.sub.4HCO.sub.3); B: MeCN; A %-B %=20%-45%, 20 minutes] to give compound 3-4a (R.sub.pR.sub.p or S.sub.pR.sub.p diastereoisomer, 220 mg, 208 umol) and compound 3-4b (S.sub.pR.sub.p or R.sub.pR.sub.p diastereoisomer, 220 mg, 208 umol) as white solids.

Step 5

[0449] ##STR00065##

[0450] Compound 3-5

[0451] To a solution of compound 3-4a (100 mg, 105 umol) in MeOH (3.0 mL) was added NH.sub.4F (38.9 mg, 1.05 mmol, 10 eq) and the resulting mixture was stirred at 60 C. for 12 h. The reaction mixture was then allowed to reach RT and concentrated under reduced pressure. The residue was taken up in H.sub.2O (1 mL) at 40 C., cooled to 5 C. and kept stirring for 30 minutes, then filtered and the filter cake was collected to give compound 3-5a (20.0 mg, 26.3 umol,) as a white solid.

[0452] Reaction of compound 3-4b in a similar manner afforded compound 3-5b (33.0 mg, 45.6 umol) as a white solid.

Step 6

[0453] ##STR00066##

(1R,6R,8R,9R,10R,12R,15R,17R,18R)-17-(4-amino-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-8-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dimercapto-2,4,7,11,13,16-hexaoxa-3.SUP.5., 12.SUP.5.-diphosphatricyclo[13.2.1.0.SUP.6,10.]octadecane-3,12-dione, disodium salt (Example 3a and Example 3b)

[0454] To a solution of compound 3-5a (20.0 mg, 27.6 umol) in H.sub.2O (5 mL) was added DOWEX 50WX8 (Na.sup.+ form; 300 mg) and the mixture was stirred at RT for 3 h. The reaction was then filtered, and the filtrate was lyophilized to give Example 3a as a white solid (R.sub.pR.sub.p or S.sub.pR.sub.p, 18.0 mg, 24.5 umol).

[0455] MS(ES.sup.+) C.sub.21H.sub.26N.sub.9O.sub.10P.sub.2S.sub.2 requires: 690, found: 690.0 [M+H].sup.+; .sup.1H-NMR (400 MHz, CD.sub.3OD) ppm 8.33 (s, 1H), 8.20 (s, 1H), 8.05 (s, 1H), 7.84 (d, J=4.0 Hz 1H), 6.62 (d, J=3.6 Hz, 1H), 6.51 (d, J=8.4 Hz, 1H), 6.12 (d, J=6.8 Hz, 1H), 5.59-5.60 (m, 1H), 5.35-5.59 (m, 1H), 5.21-5.24 (m, 1H), 4.25-4.45 (m, 4H), 3.99 (d, J=12.0 Hz, 1H), 3.90-3.92 (m, 1H); .sup.31P NMR (162 MHz, CD.sub.3OD) ppm 60.36, 60.28; R.sub.t=1.497 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

[0456] Reaction of compound 3-5b in a similar manner gave Example 3b (S.sub.pR.sub.p or R.sub.pR.sub.p, 33.0 mg, 44.9 umol) as a white solid.

[0457] MS(ES.sup.+) C.sub.21H.sub.26N.sub.9O.sub.10P.sub.2S.sub.2 requires: 690, found: 690.0 [M+H].sup.+; .sup.1H-NMR (400 MHz, CD.sub.3OD) ppm 8.34 (s, 1H), 8.21 (s, 1H), 8.07 (s, 1H), 7.97 (d, J=3.6 Hz 1H), 6.72 (d, J=4 Hz, 1H), 6.56 (d, J=8.0 Hz, 1H), 6.12 (d, J=3.2 Hz, 1H), 5.20-5.31 (m, 2H), 4.98-5.01 (m, 1H), 4.71 (d, J=4 Hz, 1H), 4.33-4.49 (m, 1H), 4.25-4.30 (m, 3H), 4.07-4.12 (m, 1H); .sup.31P NMR (162 MHz, CD.sub.3OD) ppm 57.30, 53.90; R.sub.t=1.647 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

Example 4a, 4b, 4c and 4b

Cyclic dinucleotides RR-CD-A-2Cl-A, RS-CD-A-2Cl-A, SS-CD-A-2Cl-A, and SR-CD-A-2 Cl-A; dithio-[R.SUB.p., R.SUB.p.]-cyclic-[A(2,5)p-2Cl-A(3,5)p], dithio-[R.SUB.p., S.SUB.p.]-cyclic-[A(2,5)p-2Cl-A(3,5)p], dithio-[S.SUB.p., S.SUB.p.]-cyclic-[A(2,5)p-2Cl-A(3,5)p]dithio-[S.SUB.p., R.SUB.p.]-cyclic-[A(2,5)p-2Cl-A (3,5)p]

[0458] ##STR00067##

Step 1

[0459] ##STR00068##

[0460] Compound 4-1.

[0461] To a solution of Int F (3.00 g, 6.61 mmol, 1.0 eq) in DCM (60 mL) was added TEA (0.334 g, 3.31 mmol, 0.46 uL, 0.5 eq). The volatiles were removed under reduced pressure, pyridine-TFA (2.55 g, 13.2 mmol, 2.0 eq) was added to the residue, and the mixture was co-evaporated three times with anhydrous CH.sub.3CN (40 mL). The residue was dissolved in anhydrous CH.sub.3CN (30 mL) and stirred with 3A molecular sieves (3.00 g, 6.61 mmol, 1.0 eq) for five minutes. In a separate vessel Int D (6.53 g, 6.61 mmol, 1.0 eq) was co-evaporated three times with anhydrous CH.sub.3CN (20 mL), then dissolved in anhydrous CH.sub.3CN (30 mL). The resulting solution of Int D was added to the mixture of Int F, pyridine-TFA and 3 molecular sieves, followed by DDTT (1.63 g, 7.93 mmol, 1.2 eq), and the resulting mixture was stirred at 25 C. for 30 min. The mixture was then concentrated under reduced pressure to give Compound 4-1 (9.0 g) as a yellow solid which was used for the next step without further purification.

##STR00069##

Step 2

[0462] Compound 4-2.

[0463] To a solution of Compound 4-1 (9.0 g, 6.56 mmol, 1.0 eq) in DCM (90 mL) was added Cl.sub.2CHCOOH acid (2.7 mL). The mixture was stirred at 25 C. for 1 hr, and triethylsilane (4.57 g, 39.3 mmol, 6.28 mL, 6.0 eq) was added followed by pyridine (45.0 mL). The resulting mixture was stirred at RT for 30 minutes, then concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (DCM:MeOH=10:1 to 3:1) to give Compound 4-2 (2.12 g, 1.98 mmol, 30% yield) as a light yellow solid. MS(ES.sup.+) C.sub.43H.sub.50ClN.sub.11O.sub.12P.sub.2SSi requires: 1069, found: 1070 [M+H].sup.+.

Step 3

[0464] ##STR00070##

[0465] Compound 4-3a and Compound 4-3b.

[0466] Compound 4-2 (2.12 g, 1.98 mmol, 1.0 eq) was co-evaporated three times with anhydrous Pyridine (40.0 mL), dissolved in anhydrous Pyridine (40.0 mL) and the solution was cooled to 0 C. DMOCP (1.28 g, 6.93 mmol, 3.5 eq) was added, and the mixture was stirred for 0.5 h at RT. 3H-1,2-Benzodithiol-3-one 1,1-dioxide (0.595 g, 2.97 mmol, 1.5 eq) was then added, and the resulting mixture and stirred at RT for further 30 minutes. The reaction mixture was quenched by addition of 3.4% aq. NaHCO.sub.3 (600 mL), and then extracted with EtOAc (2300 mL). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by SiO.sub.2 gel chromatography (CH.sub.2Cl.sub.2/MeOH=30/1 to 10/1) to give compound 4-3a (0.470 g, 0.348 mmol, 17% yield) and compound 4-3b (0.380 g, 0.28 mmol, 14% yield), each one as a mixture of diastereoisomers of undefined stereochemistry at the phosphorothioate centers.

Step 4

[0467] ##STR00071##

[0468] Compound 4-4a, 4-4b, 4-4c, 4-4d.

[0469] To a solution of Compound 4-3a (0.47 g, 0.43 mmol, 1.0 eq) in MeOH (3 mL) was added NH.sub.4OH (4.2 g, 519 mmol, 277 eq). The mixture was stirred at 50 C. for 16 h in a pressure safe steel vessel, then cooled to RT and concentrated under reduced pressure. The residue was purified by prep-HPLC [Agela Durashell C18 15025 5 um; mobile phase: A: H.sub.2O (10 mM NH.sub.4HCO.sub.3); B: MeCN; A %-B %=10%-40%, 10.5 minutes] to give two stereoisomer products of undefined stereochemistry at the phosphorothioate centers; compound 4-4a (93.0 mg, 0.11 mmol, 25% yield) and compound 4-4c (105 mg, 0.13 mmol, 29% yield), both as white solids.

Compound 4-3b (0.380 g, 0.28 mmol) was reacted in the same manner to give compounds 4-4b (82.0 mg, 99.6 umol, 28% yield) and 4-4d (82.0 mg, 99.6 umol, 28% yield) as two stereoisomers of undefined stereochemistry at the phosphorothioate centers, both as white solids.

Step 5

[0470] ##STR00072##

[0471] Compound 4-5a, 4-5b, 4-5c, 4-5d.

[0472] To a solution of compound 4-4a (93.0 mg, 0.11 mmol, 1.0 eq) in MeOH (3.0 mL) was added NH.sub.4F (80 mg, 2.17 mmol, 20.0 eq) and the resulting mixture was stirred at 65 C. for 16 h. The reaction mixture was then allowed to cool to RT and concentrated under reduced pressure. The residue was purified by prep-HPLC [Agela Durashell C18 15025 5 um; mobile phase: A: H.sub.2O (10 mM NH.sub.4HCO.sub.3); B: MeCN; A %-B %=10%-40%, 10.5 minutes] to give compound 4-5a (50.0 mg, 65.1 umol, 60% yield) as a white solid; Compounds 4-4b, 4-4c and 4-4d were reacted in the same manner to give the following compounds: 4-5b (40.0 mg, 56.4 umol, 56% yield); 4-5c (58.0 mg, 78.1 umol, 64% yield); and 4-5d (20.0 mg, 22.6 umol, 23% yield).

Step 6

[0473] ##STR00073##

Example 4a, 4b, 4c and 4d

[0474] To a solution of compound 4-5a (50.0 mg, 67.3 umol, 1.0 eq) in H.sub.2O (20.0 mL) was added Dowex-50WX8 (Na.sup.+ form; 500 mg) and the mixture was stirred at RT for 4 h. The reaction was then filtered, and the filtrate was lyophilized to give Example 4a (36.5 mg, 46.6 umol, 69% yield) as a white solid; single stereoisomer, undefined stereochemistry at the phosphorothioate centers; MS(ES.sup.+) C.sub.20H.sub.23ClN.sub.10O.sub.9P.sub.2S.sub.2 requires: 708, found: 709 [M+H].sup.+; .sup.1H-NMR (400 MHz D.sub.2O) ppm 8.68 (s, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 8.01 (s, 1H), 6.28 (d, J=2.0 Hz, 1H), 6.17 (d, J=8.4 Hz, 1H), 5.29-5.40 (m, 2H), 5.18-5.26 (m, 1H), 4.62 (d, J=4.0 Hz, 1H), 4.57 (br s, 1H), 4.42-4.51 (m, 2H), 4.14-4.25 (m, 2H), 4.09 (br d, J=12.8 Hz, 1H); .sup.31P NMR (162 MHz, D.sub.2O) ppm 55.69, 54.83; R.sub.t=1.64 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

[0475] Compounds 4-5b, 4-5c and 4-5d were reacted in the same manner to give the following compounds, all as single stereoisomers, with undefined stereochemistry at the phosphorothioate centers:

[0476] Example 4b (40.0 mg, 48.2 umol, 89.6% yield); MS(ES.sup.+) C.sub.20H.sub.23ClN.sub.10O.sub.9P.sub.2S.sub.2 requires: 708, found: 709 [M+H].sup.+; .sup.1H-NMR (400 MHz D.sub.2O) ppm 8.42 (s, 1H), 8.16 (s, 1H), 8.04 (s, 1H), 7.94 (s, 1H), 6.26 (d, J=3.1 Hz, 1H), 6.20 (d, J=8.3 Hz, 1H), 5.33-5.43 (m, 2H), 5.09-5.13 (m, 1H), 4.55-4.61 (m, 2H), 4.42-4.49 (m, 3H), 4.34-4.41 (m, 1H), 4.05-4.14 (m, 2H); .sup.31P NMR (162 MHz, D.sub.2O) ppm 56.58, 54.75; R.sub.t=1.74 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

[0477] Example 4c (35.2 mg, 46.6 umol, 59.7% yield), MS(ES.sup.+) C.sub.20H.sub.23ClN.sub.10O.sub.9P.sub.2S.sub.2 requires: 708, found: 709 [M+H].sup.+; .sup.1H-NMR (400 MHz D.sub.2O) ppm 8.65 (s, 1H), 8.19 (s, 1H), 8.08 (s, 1H), 8.05 (s, 1H), 6.37 (s, 1H), 6.17 (d, J=8.3 Hz, 1H), 5.42 (d, J=4.8 Hz, 1H), 5.33 (m, 1H), 5.08-5.16 (m, 1H), 4.93 (d, J=3.9 Hz, 1H), 4.62 (br d, J=9.0 Hz, 1H), 4.44-4.51 (m, 2H), 4.27 (m, 1H), 4.18 (br s, 2H); .sup.31P NMR (162 MHz, D.sub.2O) ppm 53.99, 51.92; R.sub.t=2.09 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

[0478] Example 4d (17.0 mg, 24.0 umol, 89.1% yield), MS(ES.sup.+) C.sub.20H.sub.23ClN.sub.10O.sub.9P.sub.2S.sub.2 requires: 708, found: 709 [M+H].sup.+; .sup.1H-NMR (400 MHz D.sub.2O) ppm 8.34 (s, 1H), 8.10 (s, 1H), 8.04 (s, 1H), 7.86 (s, 1H), 6.30 (s, 1H), 6.14 (d, J=8.4 Hz, 1H), 5.36 (m, 1H), 5.24 (m, 1H), 5.05 (d, J=4.4 Hz, 1H), 4.80 (d, J=4.0 Hz, 1H), 4.55 (br d, J=8.0 Hz, 1H), 4.36-4.44 (m, 2H), 4.32 (ddd, J=12.0, 6.4, 2.0 Hz, 1H), 4.20 (m, 1H), 4.02 (dd, J=11.2, 3.6 Hz, 1H); .sup.31P NMR (162 MHz, D.sub.2O) ppm 54.64, 52.01; R.sub.t=2.03 minutes [Waters XBridge Shield RP18 2.1*50 mm, 5 um; mobile phase: A: H.sub.2O+10 mM NH.sub.4HCO.sub.3; B: MeCN; A %-B %=0%-30%, 5.2 minutes].

[0479] The following compounds, or a salt, ester, prodrug, or tautomer thereof, can generally be made using the methods described above. It is expected that these compounds when made will have activity similar to those that have been prepared.

##STR00074##

Biological Activity Assays

THP-1 Dual Assay

[0480] The INVIVOGEN THP-1 Dual assay (catalog code: thpd-nfis) was used to evaluate the listed compounds as agonist of the STING receptor. The THP-1 Dual cells are derived from the human monocytic cell line THP-1 by stable integration of two inducible reporter constructs. This assay enables simultaneous study of the two main signaling pathways for STING: (a) the NF-B pathway, by monitoring the activity of secreted embryonic alkaline phosphatase (SEAP); and (b) the Interferon regulatory factor (IRF) pathway, by assessing the activity of a secreted luciferase (Lucia).

[0481] The procedure as set forth by the manufacturer was followed, with the following modifications: (1) plates are incubated 18 h after addition of cell suspension, and (2) the optional differentiation step is not employed.

Tables 1 and 2. Biological Activity

[0482]

TABLE-US-00001 TABLE 1 Fold induction of IRF3 activation in THP-1 Dual cells upon treatment with increasing concentrations of STING agonist. 0.1 0.5 1 5 10 mg/mL mg/mL mg/mL mg/mL mg/mL 3,5-c-di-GMP 1.07 1.26 1.32 2.87 5.93 ML-RS-CDA* 1.31 11.55 56.72 144.86 141.76 ML-RR-CDA** 2.65 4.57 8.91 148.89 166.88 Example 1a 1.14 1.84 9.77 112.64 157.93 Example 1b 0.99 1.88 3.52 25.91 87.63 Example 2a 27.89 138.29 148.06 147.12 143.02 Example 2b 16.46 95.25 156.38 159.53 157.10 Example 3a 1.41 0.98 2.07 3.04 4.29 Example 3b 1.38 1.48 1.56 5.05 6.70 Example 4d 1.99 nd 5.89 nd 22.37 *= dithio-[R.sub.p,S.sub.p]-cyclic-[A(2,5)p-A(3,5)p]; **= dithio-[R.sub.p,R.sub.p]-cyclic-[A(2,5)p-A(3,5)p]; prep'd as in WO 2014/189805. Examples 4a-4c did not show significant activity.

TABLE-US-00002 TABLE 2 Fold induction of NF-B activation THP-1 Dual cells upon treatment with increasing concentrations of STING agonist. 0.1 0.5 1 5 10 mg/mL mg/mL mg/mL mg/mL mg/mL 3,5-c-di-GMP 0.99 0.98 0.97 1.02 1.10 ML-RS-CDA* 0.99 1.05 1.42 17.03 20.05 ML-RR-CDA** 1.02 1.09 1.17 9.06 16.89 Example 1a 1.02 1.04 1.07 2.77 7.30 Example 1b 0.97 0.99 1.01 1.29 3.71 Example 2a 1.14 5.73 11.47 12.81 11.52 Example 2b 1.09 1.70 4.26 13.22 13.68 Example 3a 0.99 0.96 0.99 1.01 1.04 Example 3b 0.98 0.99 1.00 1.08 1.14 Example 4d 0.21 nd 0.24 nd 0.36 *= dithio-[R.sub.p,S.sub.p]-cyclic-[A(2,5)p-A(3,5)p]; **= dithio-[R.sub.p,R.sub.p]-cyclic-[A(2,5)p-A(3,5)p]; prep'd as in WO 2014/189805. Examples 4a-4c did not show significant activity.

Mouse Ductal Pancreatic Cancer Assay

[0483] The procedure of Boj et al. (Cell 2015, 160, 324-338) was followed, which employs murine organoids. 2.510.sup.5 MT4-2D cells were injected subcutaneously on the right flank of male 6 week old C57BL/6J mice. 5 ug of the indicated STING agonist was injected intra-tumorally on day 15 in a volume of 50 ul.

TABLE-US-00003 TABLE 3 In Vivo Activity Example 2b Days Post ML-RR-CDA Example 2a (Tumor Vol, Challenge (Tumor Vol, mm.sup.3) (Tumor Vol, mm.sup.3) mm.sup.3) 0 0.0 0.0 0.0 4 19.0 29.2 28.4 6 56.3 64.3 53.9 8 81.9 84.3 66.5 10 84.5 103.3 87.4 12 107.3 125.9 98.3 14 110.9 113.8 106.6 15 125.6 132.1 114.7 16 193.2 134.0 130.8 17 131.1 122.7 128.3 18 117.0 82.8 90.4 20 141.9 72.8 115.3 22 147.2 91.6 64.9 25 154.0 124.0 70.4 27 241.4 142.0 80.7 29 198.5 163.1 99.6 32 250.1 217.7 102.6

[0484] All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein in their entireties. Where any inconsistencies arise, material literally disclosed herein controls.

[0485] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.