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, 33006-Oviedo (Asturias), Spain b Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA c Rasayan Inc., 2802 Crystal Ridge Road, Encinitas, CA 92024-6615, 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 12... S3 13 C NMR of 12... S4 DEPT NMR of 12... S5 1 H NMR of 13... S6 13 C NMR of 13... S7 DEPT NMR of 13... S8 1 H NMR of 14... S9 13 C NMR of 14... S10 DEPT NMR of 14... S11 1 H NMR of 15... S12 13 C NMR of 15... 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 26... S45 13 C NMR of 26... S46 COSY NMR of 26... S47
HSQC NMR of 26... S48 HMBC NMR of 26... S49 NOESY NMR of 26... S50 1 H NMR of 27... S51 13 C NMR of 27... S52 COSY NMR of 27... S53 HSQC NMR of 27... S54 HMBC NMR of 27... S55 HMBC NMR of 27... S56 NOESY NMR of 27... S57 1 H NMR of 28... S58 13 C NMR of 28... S59 COSY NMR of 28... S60 HSQC NMR of 28... S61 HMBC NMR of 28... S62 NOESY NMR of 28... S63 1 H NMR of 29... S64 13 C NMR of 29... S65 COSY NMR of 29... S66 HSQC NMR of 29... S67 HMBC NMR of 29... S68 NOESY NMR of 29... S69 1 H NMR of 30... S70 13 C NMR of 30... S71 1 H NMR of 31... S72 13 C NMR of 31... S73 DEPT NMR of 31... S74 1 H NMR of 32... S75 13 C NMR of 32... S76 1 H NMR of 33... S77 13 C NMR of 33... S78 DEPT NMR of 33... 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
1 H NMR (CDCl 3, 400.13 MHz) S3
13 C NMR (CDCl 3, 100.6 MHz) S4
DEPT NMR (CDCl 3, 100.6 MHz) S5
1 H NMR (CDCl 3, 400.13 MHz) S6
13 C NMR (CDCl 3, 100.6 MHz) S7
DEPT NMR (CDCl 3, 100.6 MHz) S8
1 H NMR (CDCl 3, 400.13 MHz) S9
13 C NMR (CDCl 3, 100.6 MHz) S10
DEPT NMR (CDCl 3, 100.6 MHz) S11
1 H NMR (acetone-d 6, 400.13 MHz) S12
13 C NMR (CDCl 3, 100.6 MHz) S13
1 H NMR (CDCl 3, 600.15 MHz) S14
13 C NMR (CDCl 3, 600.15 MHz) S15
COSY (CDCl 3, 600.15 MHz) S16
HSQC (CDCl 3, 600.15 MHz) S17
NOESY (CDCl 3, 600.15 MHz) S18
NOESY (CDCl 3, 600.15 MHz) H6 H5 H4 H5 H5 H3 H1 H3 H5 H5 H4 Cross peak H5 H1 H6 S19
1 H NMR (acetone-d 6, 300.13 MHz) S20
13 C NMR (acetone-d 6, 75.5 MHz) S21
COSY (acetone-d 6, 300.13 MHz) S22
HSQC (acetone-d 6, 300.13 MHz) S23
HMBC (acetone-d 6, 300.13 MHz) S24
NOESY (acetone-d 6, 300.13 MHz) S25
NOESY (acetone-d 6, 300.13 MHz) H6 H1 H5 OH H4 H5 H5 H3 H3 Cross peak H5 H4 H5 OH Cross peak H5 H1 H6 S26
1 H NMR (CDCl 3, 400.13 MHz) S27
13 C NMR (CDCl 3, 100.6 MHz) S28
1 H NMR (MeOH-d 4, 300.13 MHz) S29
13 C NMR (MeOH-d 4, 75.5 MHz) S30
1 H NMR (acetone-d 6, 300.13 MHz) S31
13 C NMR (acetone-d 6, 75.5 MHz) S32
COSY (acetone-d 6, 300.13 MHz) S33
HSQC (acetone-d 6, 300.13 MHz) S34
HMBC (acetone-d 6, 300.13 MHz) S35
NOESY (acetone-d 6, 300.13 MHz) S36
1 H NMR (acetone-d 6, 300.13 MHz) S37
13 C NMR (acetone-d 6, 75.5 MHz) S38
COSY (acetone-d 6, 300.13 MHz) S39
HSQC (acetone-d 6, 300.13 MHz) S40
HMBC (acetone-d 6, 300.13 MHz) S41
NOESY (acetone-d 6, 400.13 MHz) S42
1 H NMR (MeOH-d 4, 300.13 MHz) S43
13 C NMR (MeOH-d 4, 75.5 MHz) S44
1 H NMR (THF-d 8, 400.13 MHz) S45
13 C NMR (THF-d 8, 75.5 MHz) S46
COSY (THF-d 8, 400.13 MHz) S47
HSQC (THF-d 8, 400.13 MHz) S48
HMBC (THF-d 8, 400.13 MHz) S49
NOESY (THF-d 8, 400.13 MHz) S50
1 H NMR (CDCl 3, 300.13 MHz) S51
13 C NMR (CDCl 3, 75.5 MHz) S52
COSY (CDCl 3, 300.13 MHz) S53
HSQC (CDCl 3, 300.13 MHz) S54
HMBC (CDCl 3, 300.13 MHz) S55
H2 HMBC (CDCl 3, 300.13 MHz) H8 H1 C2 C5 Cross peaks Cross peak C8 C2 C4 C6 C=O S56
NOESY (CDCl 3, 300.13 MHz) S57
1 H NMR (CDCl 3, 400.13 MHz) S58
13 C NMR (CDCl 3, 100.6 MHz) S59
COSY (CDCl 3, 400.13 MHz) S60
HSQC (CDCl 3, 400.13 MHz) S61
HMBC (CDCl 3, 400.13 MHz) S62
NOESY (CDCl 3, 400.13 MHz) S63
1 H NMR (CDCl 3, 400.13 MHz) S64
13 C NMR (CDCl 3, 100.6 MHz) S65
COSY (CDCl 3, 400.13 MHz) S66
HSQC (CDCl 3, 400.13 MHz) S67
HMBC (CDCl 3, 400.13 MHz) S68
NOESY (CDCl 3, 400.13 MHz) S69
1 H NMR (THF-d 8, 400.13 MHz) S70
13 C NMR (THF-d 8, 100.6 MHz) S71
1 H NMR (CDCl 3, 400.13 MHz) S72
13 C NMR (CDCl 3, 75.5 MHz) S73
DEPT NMR (CDCl 3, 75.5 MHz) S74
1 H NMR (THF-d 8, 400.13 MHz) / S75
13 C NMR (MeOH-d 4, 75.5 MHz) S76
1 H NMR (MeOH-d 4, 300.13 MHz) S77
13 C NMR (MeOH-d 4, 75.5 MHz) S78
DEPT NMR (MeOH-d 4, 75.5 MHz) S79
-> 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
-> 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
-> HPLC separation for the four anomers 22-25 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