Synthesis, evaluation of anti-hiv-1 and anti-hcv activity of novel 2,3 -dideoxy- 2,2 -difluoro-4 -azanucleosides

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1 Synthesis, evaluation of anti-hiv-1 and anti-hcv activity of novel 2,3 -dideoxy- 2,2 -difluoro-4 -azanucleosides Saúl Martínez-Montero, a,b Susana Fernández, a Yogesh S. Sanghvi, c Emmanuel A. Theodorakis,* b Mervi A. Detorio, d Tamara R. McBrayer, e Tony Whitaker, e Raymond F. Schinazi, d Vicente Gotor,* a and Miguel Ferrero* a a Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, Oviedo (Asturias), Spain b Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA , USA c Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA , USA d Center for AIDS Research, Department of Pediatrics, Emory University School of Medicine, and the Veterans Affairs Medical Center, 1670 Clairmont Road, Decatur, Georgia 30033, USA. e RFS Pharma, LLC, 1860 Montreal Road, Tucker, Georgia 30084, USA. mferrero@uniovi.es, vgs@uniovi.es, etheodor@ucsd.edu SUPPORTING INFORMATION (page 1 of 82 pages) Table of Contents 1. Copies of 1 H, 13 C, DEPT, and 2D NMR spectra of compounds 12 33: 1 H NMR of S3 13 C NMR of S4 DEPT NMR of S5 1 H NMR of S6 13 C NMR of S7 DEPT NMR of S8 1 H NMR of S9 13 C NMR of S10 DEPT NMR of S11 1 H NMR of S12 13 C NMR of S13 1 H NMR of 18a... S14 13 C NMR of 18a... S15 COSY NMR of 18a... S16 HSQC NMR of 18a... S17 NOESY NMR of 18a... S18 NOESY NMR of 18a... S19 1 H NMR of 19a... S20 13 C NMR of 19a... S21 COSY NMR of 19a... S22 HSQC NMR of 19a... S23 HMBC NMR of 19a... S24 NOESY NMR of 19a... S25 NOESY NMR of 19a... S26 1 H NMR of 18b/19b... S27 13 C NMR of 18b/19b... S28 1 H NMR of 18c/19c... S29 13 C NMR of 18c/19c... S30 1 H NMR of 18d... S31 13 C NMR of 18d... S32 S1 COSY NMR of 18d... S33 HSQC NMR of 18d... S34 HMBC NMR of 18d... S35 NOESY NMR of 18d... S36 1 H NMR of 19d... S37 13 C NMR of 19d... S38 COSY NMR of 19d... S39 HSQC NMR of 19d... S40 HMBC NMR of 19d... S41 NOESY NMR of 19d... S42 1 H NMR of 20c/21c... S43 13 C NMR of 20c/21c... S44 1 H NMR of S45 13 C NMR of S46 COSY NMR of S47

2 HSQC NMR of S48 HMBC NMR of S49 NOESY NMR of S50 1 H NMR of S51 13 C NMR of S52 COSY NMR of S53 HSQC NMR of S54 HMBC NMR of S55 HMBC NMR of S56 NOESY NMR of S57 1 H NMR of S58 13 C NMR of S59 COSY NMR of S60 HSQC NMR of S61 HMBC NMR of S62 NOESY NMR of S63 1 H NMR of S64 13 C NMR of S65 COSY NMR of S66 HSQC NMR of S67 HMBC NMR of S68 NOESY NMR of S69 1 H NMR of S70 13 C NMR of S71 1 H NMR of S72 13 C NMR of S73 DEPT NMR of S74 1 H NMR of S75 13 C NMR of S76 1 H NMR of S77 13 C NMR of S78 DEPT NMR of S79 2. HPLC Chromatograms on Crudes: a) After glycosylation of 15 with uracil. Mixture of 16a/17a... S80 b) Mixture of anomers 18b/19b after TBDMS deprotection... S81 c) After glycosylation of 15 with cytidine. Mixture of 16c/17c... S81 d) After glycosylation of 15 with 5-fluorouracil. Mixture of 16d/17d... S82 e) After glycosylation of 15 with 6-chloropurine... S82 S2

3 1 H NMR (CDCl 3, MHz) S3

4 13 C NMR (CDCl 3, MHz) S4

5 DEPT NMR (CDCl 3, MHz) S5

6 1 H NMR (CDCl 3, MHz) S6

7 13 C NMR (CDCl 3, MHz) S7

8 DEPT NMR (CDCl 3, MHz) S8

9 1 H NMR (CDCl 3, MHz) S9

10 13 C NMR (CDCl 3, MHz) S10

11 DEPT NMR (CDCl 3, MHz) S11

12 1 H NMR (acetone-d 6, MHz) S12

13 13 C NMR (CDCl 3, MHz) S13

14 1 H NMR (CDCl 3, MHz) S14

15 13 C NMR (CDCl 3, MHz) S15

16 COSY (CDCl 3, MHz) S16

17 HSQC (CDCl 3, MHz) S17

18 NOESY (CDCl 3, MHz) S18

19 NOESY (CDCl 3, MHz) H6 H5 H4 H5 H5 H3 H1 H3 H5 H5 H4 Cross peak H5 H1 H6 S19

20 1 H NMR (acetone-d 6, MHz) S20

21 13 C NMR (acetone-d 6, 75.5 MHz) S21

22 COSY (acetone-d 6, MHz) S22

23 HSQC (acetone-d 6, MHz) S23

24 HMBC (acetone-d 6, MHz) S24

25 NOESY (acetone-d 6, MHz) S25

26 NOESY (acetone-d 6, MHz) H6 H1 H5 OH H4 H5 H5 H3 H3 Cross peak H5 H4 H5 OH Cross peak H5 H1 H6 S26

27 1 H NMR (CDCl 3, MHz) S27

28 13 C NMR (CDCl 3, MHz) S28

29 1 H NMR (MeOH-d 4, MHz) S29

30 13 C NMR (MeOH-d 4, 75.5 MHz) S30

31 1 H NMR (acetone-d 6, MHz) S31

32 13 C NMR (acetone-d 6, 75.5 MHz) S32

33 COSY (acetone-d 6, MHz) S33

34 HSQC (acetone-d 6, MHz) S34

35 HMBC (acetone-d 6, MHz) S35

36 NOESY (acetone-d 6, MHz) S36

37 1 H NMR (acetone-d 6, MHz) S37

38 13 C NMR (acetone-d 6, 75.5 MHz) S38

39 COSY (acetone-d 6, MHz) S39

40 HSQC (acetone-d 6, MHz) S40

41 HMBC (acetone-d 6, MHz) S41

42 NOESY (acetone-d 6, MHz) S42

43 1 H NMR (MeOH-d 4, MHz) S43

44 13 C NMR (MeOH-d 4, 75.5 MHz) S44

45 1 H NMR (THF-d 8, MHz) S45

46 13 C NMR (THF-d 8, 75.5 MHz) S46

47 COSY (THF-d 8, MHz) S47

48 HSQC (THF-d 8, MHz) S48

49 HMBC (THF-d 8, MHz) S49

50 NOESY (THF-d 8, MHz) S50

51 1 H NMR (CDCl 3, MHz) S51

52 13 C NMR (CDCl 3, 75.5 MHz) S52

53 COSY (CDCl 3, MHz) S53

54 HSQC (CDCl 3, MHz) S54

55 HMBC (CDCl 3, MHz) S55

56 H2 HMBC (CDCl 3, MHz) H8 H1 C2 C5 Cross peaks Cross peak C8 C2 C4 C6 C=O S56

57 NOESY (CDCl 3, MHz) S57

58 1 H NMR (CDCl 3, MHz) S58

59 13 C NMR (CDCl 3, MHz) S59

60 COSY (CDCl 3, MHz) S60

61 HSQC (CDCl 3, MHz) S61

62 HMBC (CDCl 3, MHz) S62

63 NOESY (CDCl 3, MHz) S63

64 1 H NMR (CDCl 3, MHz) S64

65 13 C NMR (CDCl 3, MHz) S65

66 COSY (CDCl 3, MHz) S66

67 HSQC (CDCl 3, MHz) S67

68 HMBC (CDCl 3, MHz) S68

69 NOESY (CDCl 3, MHz) S69

70 1 H NMR (THF-d 8, MHz) S70

71 13 C NMR (THF-d 8, MHz) S71

72 1 H NMR (CDCl 3, MHz) S72

73 13 C NMR (CDCl 3, 75.5 MHz) S73

74 DEPT NMR (CDCl 3, 75.5 MHz) S74

75 1 H NMR (THF-d 8, MHz) / S75

76 13 C NMR (MeOH-d 4, 75.5 MHz) S76

77 1 H NMR (MeOH-d 4, MHz) S77

78 13 C NMR (MeOH-d 4, 75.5 MHz) S78

79 DEPT NMR (MeOH-d 4, 75.5 MHz) S79

80 -> HPLC separation for both uracil anomers 16a/17a after the glycosylation reaction Conditions: Agilent Poroshel 120 SB, C18; mixture (A: MeCN y B: H 2 O) gradient starting at 2.5% of B to reach 95% of B in 5 min; 1 ml/min, 30 ºC. -> HPLC separation for both thymine anomers 18b/19b (no conditions were found to separate the TBDMS protected nucleosides 16b/17b). Conditions: Mediterranea column C18 (250 x 45), mixture (A: 35% MeCN; B: 65% H 2 O), 1 ml/min, 30 ºC. S80

81 -> HPLC separation for both cytidine anomers 16c/17c after the glycosylation reaction. Conditions: Agilent Poroshel 120 SB, C18; mixture (A: MeCN y B: H 2 O) gradient starting at 2.5% of B to reach 95% of B in 5 min; 1 ml/min, 30 ºC. -> HPLC separation for both 5-fluorouracil anomers 16d/17d after the glycosylation reaction. Conditions: Agilent Poroshel 120 SB, C18; mixture (A: MeCN y B: H 2 O) gradient starting at 2.5% of B to reach 95% of B in 5 min; 1 ml/min, 30 ºC. S81

82 -> HPLC separation for the four anomers after glyosilation with 6-chloropurine. Conditions: Mediterranea column C18 (250 x 45), mixture (A: MeCN y B: H 2 O) gradient starting at 40% of B to reach 100% of B in 35 min; 1 ml/min, 30 ºC. S82