Synthesis and Anti-HCV Activity of Sugar-Modified Guanosine Analogues: Discovery of AL-611 as an HCV NS5B Polymerase Inhibitor for the Treatment of Chronic Hepatitis C

Article abstract of DOI:10.1021/acs.jmedchem.0c00935

Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The current standard of care for CHC can achieve cure rates above 95%; however, the drugs in current use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a shorter treatment period is highly desirable. We report synthesis and biological evaluation of a series of 2′,3′- and 2′,4′-substituted guanosine nucleotide analogues. Their triphosphates exhibited potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 μM. In the HCV replicon assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50 values as low as 5 nM. A lead compound AL-611 showed high levels of the nucleoside 5′-triphosphate in vitro in primary human hepatocytes and in vivo in dog liver following oral administration.

Full text of DOI:10.1021/acs.jmedchem.0c00935

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Article
Synthesis and Anti-HCV Activity of Sugar-Modified
Guanosine Analogues: Discovery of AL-611 as HCV NS5B
Polymerase Inhibitor for the Treatment of Chronic Hepatitis C
Guangyi Wang, Natalia Dyatkina, Marija Prhavc, Caroline Williams, Vladimir Serebryany,
Yujian Hu, Yongfei Huang, Xiangyang Wu, Tongqian Chen, Wensheng Huang, Vivek
K Rajwamshi, Jerom Deval, Amy Fung, Zhinan Jin, Antitsa Stoycheva, Kenneth Shaw,
Kusum Gupta, Yuen Tam, Andreas Jekle, David B Smith, and Leonid Beigelman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00935 • Publication Date (Web): 20 Aug 2020
Downloaded from pubs.acs.org on August 20, 2020
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Synthesis and Anti-HCV Activity of Sugar-Modified Guanosine Analogues: Discovery of
AL-611 as HCV NS5B Polymerase Inhibitor for the Treatment of Chronic Hepatitis C
8
9
Guangyi Wang,1 Natalia Dyatkina,*1 Marija Prhavc,1 Caroline Williams,1,a Vladimir
Serebryany,1,a Yujian Hu,2 Yongfei Huang,2 Xiangyang Wu,2 Tongqian Chen,3 Wensheng Huang,3
Vivek K. Rajwanshi, 1,a Jerome Deval,1,a Amy Fung,1 Zhinan Jin,1 Antitsa Stoycheva,1,a Kenneth
Shaw,1 Kusum Gupta, 1,a Yuen Tam,1 Andreas Jekle, 1,a David B. Smith, 1,a Leonid Beigelman1,a
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1 Janssen BioPharma, Inc., 260 E. Grand Ave., South San Francisco, California, 94080, USA
2 Department of Medicinal Chemistry, WuXi AppTec, Shanghai 200131, P.R. China
3Pharmaron Beijing, Co. Ltd. No. 6, TaiHe Road, BDA, Beijing, 100176, P.R. China
Abstract: Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The
current standard of care for CHC can achieve cure rates above 95%, however the drugs in current
use are administered for a period of 8-16 weeks. A combination of safe and effective drugs with a
shorter treatment period is highly desirable. We report synthesis and biological evaluation of a
series of 2’,3’- and 2’,4’-substituted guanosine nucleotide analogues. Their triphosphates exhibited
potent inhibition of the HCV NS5B polymerase with IC50 as low as 0.13 µM. In the HCV replicon
assay, the phosphoramidate prodrugs of these analogues demonstrated excellent activity with EC50
values as low as 5 nM. A lead compound AL-611 showed high levels of the NTP in vitro in primary
human hepatocytes and in vivo in dog liver following oral administration.
N
OEt
O
P
O
O
N
O
O
N
F
N
N
H
O
HO
F
NH2
AL-611
EC50 = 0.005 µM, CC50 > 100 µM
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INTRODUCTION
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Chronic hepatitis C (CHC) is a major liver disease caused by the hepatitis C virus. The standard
of care for hepatitis C has changed rapidly with the advent of new therapies and other
developments. Combinations of sofosbuvir/velpatasvir or sofosbuvir/ledipasvir are part of the
preferred regimens in the AASLD guidelines1-3 and can achieve cure rates above 95%. Therapy
with these direct antiviral agents can cure persons infected with HCV and the treatment period is
typically 12 weeks. Drugs in chronic use may develop resistance and show more adverse effects
with time. Therefore, a combination of safe and effective drugs with a shorter treatment period is
highly desirable. A shorter treatment therapy is expected to increase both patient compliance and
treatment accessibility to patients infected by HCV. Nucleoside analogues have excellent records
as antiviral drugs4. Their triphosphate metabolites may selectively inhibit viral targets, particularly
viral polymerases, effectively preventing viral replication.5 The well-known HCV drug
sofosbuvir, a uridine analogue, is widely used in HCV therapies.2, 6 Another nucleoside drug-
candidate BMS094 (INX-189), which is a prodrug of 2’-C-methylguanosine 1, is also well known
for its very potent activity against HCV infection.7 However, the drug was halted in a clinical trial
owing to its severe toxicity.8 It was reported two more guanosine prodrugs of β-D-2′-deoxy-2′-α-
fluoro-2′-β-C-methylguanosine 2: PSI-6619, a phosphoramidate nucleotide, and PSI-93810-11, a
cyclic phosphate nucleotide. Both compounds are metabolized to the same active 5′-triphosphate
of 2. Despite the potent clinical anti-HCV activity, development of these compounds was
discontinued, with PSI-938 halted due to hepatic toxicity observed with extended dosing in the
clinic. Recently a new phosphoroamidate prodrugs AT-527 was reported.12 Its active metabolite is
also 5′-triphosphate of 2. AT-527 demonstrated potent, pan-genotypic activity against hepatitis C
virus and is now in clinical development. Incorporation of the guanosine nucleoside triphosphate
into host RNA by human mitochondrial RNA polymerase was at least partially responsible for the
toxicity.13 In fact, the HCV nucleosides that are either an approved drug (such as sofosbuvir) or
were successful in clinical trials (such as AL-33514-15 or uprifosbuvir16 ) are pyrimidine analogues.
In order to obtain guanosine derivatives with a good safety profile and high antiviral potency, we
explored a variety of sugar-modified guanosine analogues. One of our approaches was to increase
the complexity of the sugar moiety to limit off-target effects caused by interactions with host
polymerases and therefore increase the chance to identify a safe nucleoside analogue. Towards this
goal, we modified the sugar moiety by combining substituents at 2’-, 3’-, and 4’-positions. In this
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article, we report the synthesis, HCV NS5B inhibition, host polymerase inhibition, and HCV
replicon activity of a series of guanosine analogues 4-10 having 2’,3’- and 2’,4’-disubstituted sugar
moieties (Figure 1). For the lead prodrug AL-611 (42) the in vivo and in vitro NTP formation,
replicon activity, and cytotoxicity are also presented.
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N
N
N
N
N
N
O
NH
O
NH
O
NH
O
O
O
N
N
N
HO
HO
HO
F
R
HO
R
HO
R1
F
R
NH2
NH2
NH2
1
2
3
4
5
6
7
R=OH
R=F
R1=OH R=CH3
8
9
R=OH
R=F
R=Cl
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
R=Cl
10
Figure 1. Sugar modified guanosine analogues.
RESULTS AND DISCUSSION
Chemistry
Synthesis of compounds 4-7 is shown in Scheme 1. Starting nucleosides were prepared according
to known procedures: 11a,17 11b,18 11c,17,19 and 11d.20 N2-monomethoxytrytilated guanosines 11a-
d were subjected to direct iodination or were converted to the alkyl iodides via a tosyl intermediate.
Treatment of 5’-iodo nucleosides 12a-d with DBU then afforded olefins 13a-d. Stereoselective
introduction of 4’-fluorine on a nucleoside using iodine and silver fluoride was first described by
Moffatt. 21 This method results in one isomer with 4’-fluorine on alpha phase of the ribose and was
used for preparation 4’-fluoro pyridine22 and pyrimidine23 nucleosides. We used this approach for
synthesis of compounds 14a,b,d from 13a,b,d. Subsequent benzoylation and silica gel
chromatography gave 15a,b,d as single isomers. Treatment of 13c with NIS and triethylamine
trihydrofluoride resulted in mixture of stereoisomers, which were separated by HPLC. The D-ribo
isomer 14c was benzoylated to yield 15c. Treatment of 15a-d with sodium benzoate at elevated
temperature produced the perbenzoylated products 16a-d. The target nucleosides 4-7 were
obtained via global deprotection using ammonia or butylamine followed by HCl/dioxane.
Configuration at C-4’ for 4-7 was confirmed by 2D NMR enhancement spectroscopy (NOESY),
interaction between H-8 and H-5’,5” demonstrated that the 4’-F is on the alpha face of the ribose.
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The synthesis of compounds 9 and 10 is shown in Scheme 2. Starting 3-fluoro-O-methylriboside
18 was prepared as described earlier.24 Condensation of 18 with 6-chloro-2-aminopurine under
Vorbruggen conditions gave nucleoside 19 with good yield. Monomethoxytrityl protection of the
heterocyclic base and subsequent debenzoylation resulted in nucleoside 20. TEMPO-mediated
oxidation of the 2’-alcohol to the ketone provided intermediate 21 with high yield. Grignard
methylation of ketone 21 followed by fluorination with DAST afforded 2’,3’-dideoxy-2’,3’-
difluoronucleoside 22 as a single isomer, which was transformed to guanosine 9 under standard
conditions.
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Scheme 1
N
N
N
N
N
N
O
O
O
O
O
O
N
N
N
a or b
c
d or e
HO
I
NH
NH
NH
R
R
R
HO
R1
HO
R1
HO
R1
NHMMTr
NHMMTr
NHMMTr
11a
11b
11c
11d
12a
13a
13b
13c
13d
R1=OH R=CH3
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
R1=OH R=CH3
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
R1=OH R=CH3
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
12b
12c
12d
N
N
N
N
N
O
O
O
f or g
O
O
N
h
O
N
N
I
BzO
F
I
NH
NH
NH
F
F
R
R
R
N
HO
R1
BzO
R1
BzO
R1
NHMMTr
NHMMTr
NHMMTr
16a
16b
16c
16d
R1=OBz R=CH3
R1=OBz R=CCH
R1=F, R=CH3
14a
14b
14c
14d
R1=OH R=CH3
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
15a
R1=OBz R=CH3
15b
15c
15d
R1=OBz R=CCH
R1=F, R=CH3
R1=F, R=CCH
R1=F, R=CCH
N
N
N
O
O
l
j or k
HO
O
O
N
HO
F
NH
NH
F
R
R
N
N
HO
R1
HO
R1
NHMMTr
NH2
17a
17b
17c
17d
R1=OH R=CH3
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
4
5
6
7
R1=OH R=CH3
R1=OH R=CCH
R1=F, R=CH3
R1=F, R=CCH
o
Reagents and conditions: (a) i.TsCl, pyridine, 0 C, 1 h; ii. NaI, acetone, reflux, overnight, 41% for 12a,
52% for 12b, 41% for 12c; (b) PPh3, imidazole, I2, THF, rt, overnight, 90% for 12d; (c) DBU, THF, 60 oC,
.
overnight, 60% for 13a, 62% for 13b, 67% for 13c, 83% for 13d; (d) AgF, I2, DCM, rt, 3 h; (e) NEt3 3HF,
NIS, CH3CN, rt, 2 h, 16% for 14c; (f) BzCl, DMAP, Et3N, DCM, rt, overnight, 81% for 15c; (g) BzCl,
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pyridine; (h) HMPA, NaOBz, 15-crown-5, 48h, 60 oC, yield for 3 steps 15% for 16a from 13a, 6% for
16b, 11% for 16d; (j) NH3/MeOH, 49% for 2 steps for 17c; (k) n-butylamine, rt, 80% for 17a, 86% for 17b,
75% for 17d; (l) HCl/dioxane, CH3CN, 80% for 4, 73% for 5, 81% for 6, 36% for 7 (for 2 steps).
7
8
9
Scheme 2
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N
N
N
N
Cl
Cl
O
N
O
N
c
O
O
BnO
N
b
BnO
N
a
BnO
BnO
F
OBz
F
OH
NH2
F
OBz
NHTr
20
18
19
N
N
N
N
N
Cl
O
NH
Cl
O
O
N
N
O
N
d
e
BnO
HO
N
N
N
F
F
F
F
F
O
NH2
NH2
NHTr
21
22
9
N
N
Co
O
f
t-Bu
O
t-Bu
N
N
N
Cl
N
Cl
O
N
O
N
t-Bu t-Bu
g
h
24
BnO
BnO
N
N
F
F
Cl
NHAc
NHAc
23
25
N
N
N
O
NH
Cl
N
O
N
O
N
j
HO
HO
N
F
Cl
F
Cl
NH2
NHAc
10
26
Reagents and conditions: (a) BSA, TMSOTf, CH3CN, 80 oC, 16 h, 72%; (b) i. TrCl, Et3N, DMAP, DMF,
45 oC, 24 h, 81%; ii. NH3/MeOH, rt, 4h, 78%; (c) i. TEMPO, PhI(OAc)2, AcOH, DCM, rt, 16 h, 100%; (d)
i. CH3MgBr, ether, 2 h, -30 oC, 20%; ii. DAST, DCM, rt, 2 h, 22%; (e) i. NaOMe, 2-mercaptoethanol, 12
h, 65 oC; ii. BCl3, DCM, -75 oC, 0.5 h, 55% for 2 steps; (f) i. CH3Ph3PBr, LiHMDS, THF, rt, 4 h, 34%; ii.
AcOH, MeOH, 50 oC, 5 h, 60%; iii. AcCl, pyridine, DCM, rt, 2 h, 65%; (g) TsCl, PhSiH3, EtOH, 30 oC,
2.5 h, 29%; (h) BCl3, DCM, -30 oC, 3 h; (j) NaOMe, 2-mercaptoethanol, 12 h, 60 oC, 26% for 2 steps.
Ketone 21 could be converted to the exocyclic alkene 23 by treatment with potassium salt of
methylphosphonium bromide followed by subsequent detritylation and acetylation of the purine
base. Compound 23 was a subjected to a hydrochlorination reaction25 with TsCl in the presence of
Co catalyst26 24 to yield the desired 2’-Cl guanosine 25 as a single isomer. Exclusive formation of
the desired alpha-isomer of the 2’-chloride was most likely due to the steric congestion of the beta
face of the ribose. Debenzylation of 25 with BCl3 afforded 26 which was treated with NaOMe and
thioethanol to provide target guanosine 10. Stereoconfiguration at C-2’ for both 9 and 10 was
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proven by NMR. In both cases, NOE interaction between H-8 and 2’-CH3 demonstrated the methyl
to be on the ꞵ-face of the nucleoside.
Nucleosides 1,27 2,28 3,18 and 819 were synthesized according published procedures. All
nucleosides 1-10 in Figure 1 were converted to their 5’-O-triphosphates for testing in viral and
human polymerase assays.
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Scheme 3
N
N
N
N
N
N
Cl
OEt
OEt
O
O
N
N
O
N
a
b
BzO
HO
N
N
I
N
BzO
F
HO
F
HO
F
NH2
NHMMTr
NHMMTr
29
27
28
c
N
N
N
N
OEt
OEt
N
OEt
N
O
O
O
N
N
N
d
h
e
BzO
N
I
F
F
N
N
BzO
32
F
HO
F
HO
F
NHMMTr
NHMMTr
NHMMTr
30
31
g
f
N
N
N
OEt
OEt
OEt
N
O
O
N
N
O
N
HO
N
N
BzO
N
F
HO
F
N
F
N
MMTrO
F
MMTrO
F
HO
F
NHMMTr
NHMMTr
NHMMTr
35
34
33
j
N
OEt
51
52
53
R = ethyl
R = isobutyl
R = isopropyl
N
O
O
F
N
O
N
P
NH2
O
F
O
R
Reagents and conditions: (a) i. MMTrCl, AgNO3, collidine, DCM, rt, overnight, 84%; ii. NaOEt, EtOH, rt,
1 h, 98%; (b) I2, PPh3, imidazole, THF, rt, 5 h, 75%; (c) DBU, THF, 75 oC, 3 h, 37%; (d) NEt3 3HF, NIS,
.
DCM, 0 oC, 2 h, 60%; (e) i. BzCl, Py, rt, overnight; ii. NaOBz, DMF, 15-crown-5, 100 oC, 48 h, 45% (for
2 steps); (f) n-butylamine, rt, overnight, 85%; (g) i. MMTrCl, AgNO3, collidine, DCM, rt, overnight, 82%;
ii. NaOBz, DMF, 15-crown-5, 95 oC, 72 h, 65%; (h) n-butylamine, rt, overnight, 56%; (j) i. tert-BuMgCl,
alkyl phosphorodichloridate, THF, rt, 2-3 h, 12-32%; ii. HCl/dioxane, rt, 1 h, 95%.
Guanosine analogues were shown to be weakly active in the HCV replicon assay. The weak
potency is a result of poor phosphorylation at some stage of nucleoside phosphorylation cascade.29
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For testing in cell-based assays, nucleosides were converted to monophosphate prodrugs to
overcome the most discriminating the first phosphorylation step. Protection of the guanosine was
necessary to avoid functional group incompatibility during prodrug synthesis. The 6-OEt group
has been shown to be a viable guanosine prodrug motif. 9, 30
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Synthesis of the protected 6-OEt nucleosides 33 and 35 is shown in Scheme 3. The amino group
of the nucleoside 276 was monomethoxytritylated and obtained product was treated with NaOEt
to afford 28. Fluorination of 28 was achieved using the same synthetic strategy as shown in Scheme
1, via formation of olefin 30. Iodofluorination with triethylamine trihydrofluoride and NIS
provided nucleoside 31 as a single isomer in a good yield. Nucleophilic substitution of the iodo
moiety with OBz, together with simple protecting groups manipulations yielded 33 and 35 as
protected starting materials for the synthesis of prodrugs of 2’,4’-difluoro-2’-C-methyl-guanosine
6. Nucleoside 33 was converted to cyclic phosphates 51-53. Nucleoside 35 was a starting material
for preparation of phosphoroamidate prodrugs 39-50 (Figure 2). Protected 3’-O,N2-
bismonomethoxytrityl-O6-ethyl analogues of nucleosides 4, 5, and 7 were synthesized in a similar
way and the synthesis thereof described previously.19 These compounds were converted to 5’-
phosphoroamidate prodrugs 36-38 (Figure 2) using standard conditions.31 All prodrugs were
isolated as mixtures of diastereomers.
N
N
N
N
OEt
OEt
O
P
O
O
P
O
O
O
N
N
O
O
O
O
N
N
R1
F
F
N
H
R
N
H
O
O
HO
R2
HO
F
NH2
NH2
36
37
38
R1 = CCH, R2 = OH
R1 = CH3, R2 = OH
R1 = CCH, R2 = F
39
40
43
45
46
47
R = cyclohexyl
R = isopropyl
R = neopentyl
R = isobutyl
R = cyclopentyl
R = pentan-3-yl
N
OEt
N
N
OEt
N
O
O
O
N
O
O
P O
O
O
N
N
F
F
R
N
H
N
P
O
NH2
O
HO
F
F
O
R
NH2
44
48
49
50
R = isobutyl, X = CH
R = neopentyl, X = N
R = neopentyl, X = CH
R = isopropyl, X = CH
51
52
53
R = ethyl
R = isobutyl
R = isopropyl
X
Figure 2. Structure of synthesized prodrugs.
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7
8
9
Among these mixtures was a pair of P-isomeric prodrugs 41 and 42 (AL-611), which were
separated from diastereomeric mixture 40 by RP HPLC. For assignment of the stereochemistry at
the P-atom of 41 and 42 (AL-611), an alternative synthesis was performed. Nucleoside 35 was
treated with Sp-diastereomer of the phosphoramidating reagent reported by Ross et al.32 Reaction
with the 5’-hydroxyl group of 35 through selective nucleophilic displacement of pentafluorophenol
resulted in Sp-diastereomer identical to prodrug 42 by HPLC and 1H-NMR (Scheme 4). Therefore
42 (AL-611) was assigned as Sp- and 41 as Rp-diastereomer.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4
N
F
F
F
N
N
OEt
OEt
O
O
O
N
(S) P
N
O
F
O
N
O
(S) P
N
O
a
O
N
(S)
HO
F
N
(S)
O
N
F
+
H
O
O
H
O
HO
F
F
MMTrO
F
NH2
NHMMTr
42
35
54
Reagents and conditions: (a) i. tert-BuMgCl, THF, 5 oC, 1 h; ii. HCl/ CH3CN
Cyclophosphate prodrugs of guanosine analogues are known to demonstrate anti-HCV activity
when tested in vitro and in vivo.10-11 We synthesized cyclophosphates 51-53 (Figure 2) in one step
by treatment of nucleoside 33 with the corresponding phosphodichloridate in presence of tert-
BuMgCl. Subsequent deprotection and isolation by silica chromatography yielded cyclophosphate
prodrugs 51-53 as mixture of isomers in moderate yields (Scheme 3).
X-Ray Crystal Structure of Compound 42
The absolute stereochemisty of compound 42 (AL-611) was assigned by small-molecule X-ray
crystallography, from a difraction data set with resolution of 0.81 Å (Figure 3). The chiralities of
all stereocenters were confirmed as follows: P1 (S); C1 (R); C2 (R); C3 (S); C4 (S); C21 (S). Atom
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numbers of chiral centers are shown in Figure 3. Compound 42 is, unambiguosly, the Sp isomer.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. X-ray crystal structure of compound 42
Inhibition of HCV NS5B Polymerase
To determine the inhibitory effect of nucleosides on HCV NS5B polymerase, the 5’-triphosphates
of nucleosides 1-9 shown in Figure 1 were tested in an HCV polymerase assay. As shown in Table
1, most of the NTPs demonstrated good inhibitory activity against HCV NS5B polymerase with
IC50 as low as 0.064 µM. Comparison of IC50 values of 2’-C-methyl-GTP (1-TP) with its 4’-
fluorinated analogue (4-TP) demonstrated increased potency for the 4’-fluorinated nucleotide
(0.42 µM and 0.14 µM, respectively). 2’-Fluoro-2’-C-methyl-GTP (2-TP) and its 4’-fluorinated
analogues (6-TP) demonstrated similar inhibition (0.099 µM and 0.16 µM, respectively). These
data suggest that fluorination at the 4’-position does not alter inhibitory properties of compounds
on NS5B. The replacement of the 2’-methyl (4-TP and 6-TP) with a 2’-ethynyl group (5-TP and
7-TP) appeared well tolerated with IC50 values of 0.13-0.16 µM. Compound 3-TP which has a 2’-
α-chloro-2’-C-methyl substitution exhibited potent inhibition with IC50 value of 0.064 µM despite
containing the larger Cl substituent. When fluorine was introduced at the 3’-position of 8-TP, 9-
TP, and 10-TP, measured inhibitory effects were lower than of the corresponding 3’-OH
compounds 1-TP, 2-TP, and 3-TP, suggesting 3’-fluorination is not beneficial for inhibition of
NS5B.
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Table 1. Inhibition of HCV Polymerase NS5B by Guanosine Triphosphates.a
8
9
N
O
O
O
O
O
N
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HO P O P O P O
OH OH OH
NH
R1
R4
N
R3
R2
NH2
R1
R2
R3
R4
H
H
H
F
IC50 [µM]
0.42
NTP
1-TP
2-TP
3-TP
4-TP
5-TP
6-TP
7-TP
8-TP
9-TP
10-TP
CH3
CH3
CH3
CH3
CCH
CH3
CCH
CH3
CH3
CH3
OH
F
OH
OH
OH
OH
OH
OH
OH
F
0.099
0.064
0.14
Cl
OH
OH
F
F
0.13
F
0.16
F
F
0.15
OH
F
H
H
H
0.63
F
0.31
Cl
F
0.76
a IC50 indicates a concentration at which the activity of HCV NS5B polymerase is inhibited by
50%. Each IC50 value is an average of n ≥ 2 determinations.
To unravel the mechanism of action of 4’-fluorinated GTPs, 6-TP was tested in a chain termination
assay. We measured the incorporation of single nucleotides with fixed NS5B enzyme and RNA
concentrations, using a polyacrylamide gel-based method, as previously described.33 We isolated
the stable NS5B-RNA in the elongation mode and incubated this elongation complex with various
combinations of nucleotides. As shown in Figure 4, when natural GTP is missing, the 10-mer
elongation complex cannot be further elongated (lane 1 and 2). When only GTP was added to the
reaction, we observed the formation of the 11-mer product corresponding to single guanosine
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monophosphate (GMP) incorporation (lane 3). When GTP, UTP, and ATP were added together
to the reaction, we observed an expected 20-mer full-length RNA product (lane 4). Similarly,
substituting natural GTP by 3’dGTP and 6-TP supported the formation of an 11-mer product
(lanes 5 and 7, respectively) but stopped further elongation to the full-length 20-mer product in the
presence of UTP and ATP (lanes 6 and 8, respectively). Therefore, we concluded that
incorporation of 6-TP resulted in immediate chain termination.
10
11
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13
14
15
16
17
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19
20
21
22
23
24
25
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Primer extension and chain termination with 6-TP. A. reaction scheme as described in
Methods. B. reaction products shown on a sequencing gel. Lane 1, No NTP; Lane 2, 10 µM ATP
+ 10 µM UTP ; Lane 3, 10 µM GTP; Lane 4, 10 µM GTP + 10 µM ATP + 10 µM UTP; Lane 5,
100 µM 3’dGTP; Lane 6, 100 µM 3’dGTP + 10 µM ATP + 10 µM UTP; Lane 7, 100 µM 6-TP;
Lane 8, 100 µM 6-TP + 10 µM ATP + 10 µM UTP.
Inhibition of Human Polymerases
Host polymerases have been generally regarded as the primary off-targets for nucleoside
analogues.34 It was reported association between HCV nucleoside inhibitors that interact with
human mitochondrial DNA (hPolγ) and RNA (HMRP) polymerases and the observation of adverse
events.13, 35 For assessment of the selectivity between viral and human polymerase inhibitions,
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selected NTPs from Table 1 were tested for inhibition of human DNA polymerases α, β, and  as
well as human RNA pol II.36 The results are listed in Table 2. None of the NTPs tested inhibited
9, 12
human DNA pol-β and human RNA pol II. However, 2-TP
and 6-TP showed appreciable
7
8
9
inhibition of human DNA pol-α with 79% and 73% inhibition, respectively, at 100 M. The
inhibition by 2-TP and 6-TP might be caused by the replacement of 2’-OH with fluorine. Human
DNA pol-α tolerated 2’-α-F nucleotide to a certain degree. Inhibition of human DNA pol-α by 2-
TP and 6-TP and inhibition of human DNA pol- by 8-TP should be noted. Inhibition of human
DNA pol- by 8-TP was higher (81%), 2-TP and 6-TP didn’t demonstrate any significant
inhibition of hPolγ.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Inhibition of Human Polymerases by Nucleoside Triphosphates
hRNA pol
II
hPol-α
hPol-β
hPol-
%
NTP
IC50 [M]
% inhibition Standard
% inhibition Standard
Standard
deviation
inhibition
@ 100 uM
@ 100 uM
deviation
@ 100 uM
deviation
1-Tp 3.6
2-Tp 78.7
3-Tp 36.0
4-TP 36.0
6-TP 73.2
8-TP 16.8
8.1
2.0
4.2
2.9
0.6
11.6
15.3
14.3
21.1
20.9
16.3
8.4
11.6
12.6
5.3
7.1
9.4
8.7
2.8
1.4
4.7
0.6
>100
>100
>100
>100
>100
>100
24.6
38.4
37.1
29.8
81.0
10.8
14.9
11.4
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Incorporation of nucleotide by human mitochondrial RNA polymerase. Top, the RNA
primer and the DNA template used in this assay. Bottom, nucleotide incorporation products shown
on a sequencing gel. 100 µM nucleotide was used in each assay.
Six NTPs were tested in an assay investigating nucleotide incorporation by human mitochondrial
RNA polymerase (HMRP) (Figure 5). 1-TP (TP of INX-189’s parent nucleoside) was found to be
incorporated significantly, which may have contributed to INX-189’s toxicity. The 4’-F analogue
4-TP was able to reduce the incorporation, but the incorporation was still appreciable. When the
2’-OH was replaced by 2’-F, 2-TP 9, 12 could be incorporated, but at a much lesser extent compared
to 1-TP. After introducing 4’-F to 2-TP, the resulting 6-TP was no longer the substrate of HMRP,
as shown in Figure 5. Replacing of 2’-OH with Cl resulted in 3-TP which was significantly
incorporated into the RNA, similar to 1-TP. These results suggest that fluorine modification at the
2’ and 4’ positions disfavored the incorporation by HMRP. 8-TP (3’-F-2’-C-Me GTP) was not a
substrate of HMRP. Overall, the 6-TP tested did not show any alarming inhibition of the human
mitochondrial polymerases and low inhibition of DNA pol-α. However, the actual toxicity liability
cannot be assessed until tested in animal studies and in human trials eventually. Based on the
promising viral and human polymerase inhibition results, the nucleotide prodrugs of the 4’-F
analogue 4-7 were evaluated in cell based assays against HCV.
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HCV Replicon Activity
5
6
7
8
9
NTPs of 4’-fluorinated nucleosides 4-7 demonstrated similar inhibition of HCV polymerase
NS5B. To compare their in vitro activity in an HCV subgenomic replicon system37 we synthesized
compounds 36-39, phosphoramidate prodrugs of nucleosides 4, 5, 7, and 6.38 As shown in Table
3 2’-ethynyl derivatives 36 and 38 demonstrated low antiviral activity and high cell toxicity.
Prodrugs 37 and 39 of 2’-methyl nucleosides 4 and 6, respectively, demonstrated comparable
replicon activity and low toxicity. Considering that 6-TP exibited good inhibition of viral
polymerase and was not a substrate of HMRP we selected 2’,4’-difluoro-2’-C-methyl scaffold for
prodrug development.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. HCV Replicon Activity of Phosphoroamidate Prodrugs of Nucleosides 4,5,6, and 7
N
OEt
N
O
P
N
O
O
O
NH
R1
N
F
NH2
HO
R2
O
O
Parent
nucleoside
EC50 [µM] CC50 [µM]
Prodrug
R1
R2
OH
36
37
38
39
5
4
7
6
CCH
CH3
CCH
CH3
0.27
0.035
1.0
16.41
>100
3.26
OH
F
F
0.066
>100
EC50 indicates a concentration at which HCV replicon is inhibited by 50%. Each EC50
value is an average of ≥2 independent determinations. CC50 indicates a concentration
of 50% cell viability.
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Table 4. HCV Replicon Activity of 2’,4’-Difluoro-2’-C-Methylguanosine Phosphoroamidate
Prodrugs
7
8
9
N
OEt
N
Ar
N
OEt
N
O
P
H
N
N
O
O
N
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
F
O
N
P
F
O
NH2
O
O
F
NH2
O
R
HO
F
R
39-50
51-53
EC50
[µM]
CC50
[µM]
Prodrug
P-chirality Ar
achiral
R
SI
INX-189
39
0.348
16.42
>100
>100
>100
>100
>100
56.9
47.1
achiral
phenyl
phenyl
chiral, Rp phenyl
cyclohexyl 0.066
>1500
> 7000
> 1700
>20000
>500
3000
>2000
2700
315
40(41+42) achiral
isopropyl
isopropyl
isopropyl
neopentyl
isobutyl
0.014
0.057
0.005
0.19
41
42
43
44
45
46
47
48
49
50
51
52
chiral Sp
achiral
achiral
achiral
achiral
achiral
achiral
achiral
achiral
achiral
achiral
phenyl
phenyl
naphthalen-1-yl
phenyl
0.017
0.042
isobutyl
>100
92.9
phenyl
cyclopentyl 0.034
pentan-3-yl 0.23
phenyl
72.5
quinoline-5-yl
naphthalen-1-yl
naphthalen-1-yl
N/A
neopentyl
neopentyl
isopropyl
ethyl
0.12
0.06
0.02
0.31
1.34
>100
39.6
>800
660
80.5
4000
322
>100
>100
N/A
isobutyl
74
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53
achiral
N/A
isopropyl
0.03
>100
3000
6
7
8
9
Several phosphoramidate prodrugs of nucleoside 6 shown in Table 4 were synthesized and tested,
INX-189 was used as a positive control. Compounds 39, 40, and 43-50 were tested as mixtures of
diastereomers and showed activities ranging from 0.014 to 0.2 µM. Naphthalenyl-containing
prodrugs 44, 49, and 50 were very potent (0.02-0.06 µM) but also showed significant cytotoxicity
(56.9-80.5 µM). Increased lipophilicity of the ester moiety of 43, 47, and 48 also led to high
activity in replicon assay. Prodrug 40 was selected for further investigation based on high
selectivity index (SI>7000) and taking into consideration that its phosphate moiety was proven to
be not toxic.39 Cyclophosphate prodrugs 51-53 did not show superior properties compared with
phosphoramidates.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Two stereoisomers of 40 were separated and 41 and 42 were tested for replicon activity. Sp-isomer
42 was 10 times more active than corresponding Rp-isomer 41 (0.005 and 0.057 µM, respectively)
with SI>20000. This Sp-isomer 42 (AL-611) was selected for further evaluation.
Table 5. Eight Day Cytotoxicity (CC50 µM) of Compound 40 in Six Cell Lines
N
N
N
N
OEt
OEt
O
O
O
O
O
O
N
N
P
P
O
O
O
N
O
N
F
HO
N
N
O
H
O
H
F
HO
OH
NH2
NH2
40
INX-189
Cytotoxic Concentration Causing 50% Inhibition (CC50) μM
Compound/
Cell Line
Huh-7
> 100
HepG2
> 100
A549
> 100
HeLa
>100
U937
10
MT4
92
40
INX-189
0.35 ± 0.2 1.5 ± 0.07 7.8 ± 0.6 13.4 ± 1.7 0.17 ± 0.04 0.96 ± 0.2
Prodrug 40 did not show any appreciable cytotoxicity in Huh-7 cells. To assess the potential
cytotoxicity under more stringent conditions, this prodrug was tested in multiple cell lines for 8
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days. The well-known toxic compound INX-189 was used as a control. Compound 40
demonstrated no measurable dose-dependent decreases in cell viability (CC50 > 100 µM) across
most cell lines except for U937 and MT-4 cells where the concentration causing 50% cellular
10 and 92 µM, respectively. In all cases, the cytotoxicity of compound 40 was significantly lower
than that observed for INX-189 (Table 5).
10
11
12
13
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15
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18
19
20
21
22
23
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25
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28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In Vivo and in Vitro NTP Formation
NTP levels in the target tissue are important for in vivo efficacy of an antiviral nucleoside
polymerase inhibitor. In vitro NTP formation in appropriate cell lines may provide the first
assessment for predicting the in vivo NTP concentration. To understand difference in the extent of
in vitro activation of the individual diastereomers 41 and 42 and the isomeric mixture 40 the in
vitro NTP levels were measured in fresh primary human hepatocytes as the most relevant cell line.
As shown in Table 6, high levels of 6-TP were formed in human hepatocytes following an
incubation of 40, 41, and 42 at 50 µM extracellular concentration for 24 hours.
Table 6. NTP Formation in Vitro and in Vivo
Compound
Human Hepatocytes
Dog liver NTP
(µM) at 4h
(pmol/million cells) at 24 h
NMP
89
NTP
40
41
42
271
174
321
11.2
6.33
22.3
67.1
105
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9
NTP Time Course
1000
100
10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
20
40
Time (hr)
60
80
Figure 6. Time course of intracellular NTP in primary human hepatocytes following an incubation
with 40 at 10 µM for 2-72 h.
The experiment presented in Figure 6 was conducted to determine time-course and steady state of
NTP formation. In human hepatocytes, the steady state (equilibrium between NTP formation and
degradation) was typically reached between 2 and 16 hours (Figure 6). The decrease in NTP
formation beyond 24-48 hours is likely due to decline in function of fresh hepatocytes, a known
phenomenon. Intracellular concentrations of 6-TP were measured at multiple dose concentrations
to determine dose-linearity of the NTP formation. 6-TP Intracellular concentrations increased dose
proportionally between 0.5-10 µM of 40 incubation concentrations (Figure 7) but was not observed
at >10 µM. At incubation concentrations beyond 10 µM the NTP levels increased in less than
dose proportional manner.
Following a 24-hr incubation of 40 (~1:1 mixture of 41 and 42) in human hepatocytes, high
concentrations of NTP were formed. Once formed and after the removal of 40 from the incubation
media, NTP levels were maintained for 6 hours before degradation (Figure 8). The mean
intracellular half-life of the NTP was estimated as 11.2 ± 0.7 hours (n = 3) in human hepatocytes.
In vivo NTP formation was evaluated in beagle dogs following a single oral administration of 40,
41, and 42 at 9.69 mg/kg (5 mg/kg parent nucleoside equivalent dose). As shown in Table 6, liver
NTP concentrations were 11.2, 6.3, and 22.3 µM at 4 hours post dose of 40, 41, and 42,
respectively.
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7
8
9
900
800
700
600
500
400
300
200
100
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
10
20
30
40
50
40 Extracellular Concentration (µM)
Figure 7. Intracellular NTP concentration as a function of extracellular concentration of compound
40 in primary human hepatocytes. Data were collected from hepatocytes from three different
human donors and expressed as mean ± standard deviation. Each experiment was conducted with
24 h of incubation of 40 with hepatocytes.
1000
100
10
-10
0
10
20
30
40
50
Time (hr)
Figure 8. Intracellular NTP concentration as a function of time in primary human hepatocytes
following an initial 24 h incubation with 40 at 50 µM.
CONCLUSION
We demonstrated that guanosine nucleoside with properly modified ribose moiety can exhibit
potent HCV replicon activity and acceptable safety properties. 5’-Triphosphates of 4’-fluorinated
guanosine derivatives demonstrated potent inhibition in the HCV polymerase NS5B assay with
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IC50 values as low as 0.13 µM. The 5’-triphosphate of 2’,4’-difluoro-2’-C-methyl-guanosine (6-
TP) exhibited IC50 0.16 µM and was confirmed to be a chain terminator in a chain elongation
assay using the HCV NS5B polymerase and showed no inhibition of human DNA polymerases β
and  as well as human RNA pol II while inhibition of human DNA polymerases α was low.
Phosphoramidate prodrug of this nucleoside exhibited very potent HCV subgenomic replicon
activity with EC50 values as low as 5 nM.
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The Sp-diastereomer prodrug 42 (AL-611) was selected for preclinical toxicology studies based
on the biochemical potency of 6-TP combined with its good in vitro safety profile,
corresponding in vivo NTP formation, and cell-based replicon potency. Compound was selected
as drug candidate but given the effectiveness of already approved therapies for HCV a decision
was taken to discontinue further development.
EXPERIMENTAL SECTION
All commercially obtained solvents and reagents were used as received. All solvents used for
chemical reactions were anhydrous grade, unless specifically indicated. Structures of the target
compounds in this work were assigned by use of NMR and MS spectroscopy. The purities of
parent nucleosides and prodrugs were >95% as determined on an Agilent 1200 HPLC, Synergy 4-
µm Hydro RP 80 Å 150 × 4. 6 mm, flow 1.5 mL/min. using 0.5% HCOOH in water and 0.5%
HCOOH in CH3CN as mobile phase or 50 mM TEAA in water and 50 mM TEAA in CH3CN as
mobile phase. The purities of triphosphates were >95% determined on the same column using
linear gradient of CH3CN from 0% to 25 % in 50 mM triethylammonium acetate buffer (pH 7.5).
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1H-, F- and C-NMR spectra were recorded on a Bruker Advance III (400 MHz) or a Varian
400MR (400 MHz) NMR spectrometer. Chemical shifts are reported in parts per million (ppm, δ)
using residual solvent line as an internal reference. Splitting patterns are reported as singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), or broad singlet (br s). Coupling constants (J) are
reported in hertz (Hz). Mass spectrometric analyses for nucleosides were performed on an Agilent
1200 HPLC with Agilent 6110/6140/1956C MSD mass spectrometer using ESI as ionization.
Work-up procedures for most of chemical reactions are the same or similar, therefore, unless
specifically indicated, the work-up refers to the following procedure: the reaction mixture at 0 oC
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is quenched with water, diluted with EtOAc or dichloromethane, washed with 5% sodium
bicarbonate and then with brine, dried over anhydrous sodium sulfate, filtered, and concentrated
to dryness. Purification on silica gel refers to a flash chromatography on a silica gel column.
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5’-Deoxy-5’-iodo-2’-C-methyl-N2-(methoxytrityl)guanosine (12a). TsCl (3.43g, 17.5 mmol)
was added to the cold solution of 11a (5 g, 8.8 mmol) in pyridine (15 mL). The reaction mixture
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was stirred for 1h at 0 C and quenched with water. After usual work-up and without further
purification 5’- tosyl intermediate (6.3 g) was dissolved in acetone (60 mL) and treated with NaI
(13.2 g, 88 mmol) overnight at reflux temperature. Reaction mixture was quenched with aq.
Na2S2O3. After usual work-up and column chromatography (MeOH in DCM from 1% to 6%)
nucleoside 12a (2.4 g, 41%) was obtained as a white solid. MS, m/z 680.3 (M + 1)+. H NMR
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(CD3OD): δ 8.14 (s, 1H), 7.18-7.35 (m, 12H), 6.83-6.85 (m, 2H), 5.52 (s, 1H), 3.86-3.96 (m, 3H),
3.74 (s, 3H), 3.70-3.76 (m, 1H), 0.53 (s, 3H).
5’-Deoxy-5’-iodo-2’-C-ethynyl-N2-(methoxytrityl)guanosine (12b). Compound 12b (4 g, 52%)
as a white solid was synthesized from 6.3 g of 11b the same way as 12a. MS, m/z 690.3 (M + 1)+.
5’-Deoxy-5’-iodo-2’-fluoro-2’-C-methyl-N2-(methoxytrityl)guanosine (12c). Compound 12c
(2.4 g, 41%) as a white solid was synthesized from 5.0 g of 11c the same way as 12a. MS, m/z
682.4 (M + 1)+. H-NMR (CDCl3): δ 7.16-7.59 (m, 13H), 6.71-6.73 (m, 2H), 5.56 (d, J = 18.8
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Hz, 1H), 3.25-4.07 (m, 7H), 0.64 (d, J = 22.4 Hz, 3H).
5’-Deoxy-5’-iodo-2’-fluoro-2’-C-ethynyl-N2-(methoxytrityl)guanosine (12d). To a solution of
compound 11d (3.1 g, 5.3 mmol) in dry THF (80 mL) at 0 oC ware added PPh3 (2.8 g, 10.6 mmol)
and imidazole (720 mg, 10.6 mmol) and stirred for 30 min. To this mixture was added the solution
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of I2 (2.0 g, 8.0 mmol) in dry THF (10 mL) at 0 C and stirred at rt for 18 h. The reaction was
quenched with sat. aq. Na2SO3 (10 mL) and extracted with DCM (2 × 100 mL). The organic layer
was dried (Na2SO4) and filtered. Evaporated residue was purified by silica gel chromatography
(MeOH in DCM from 1% to 4%) to give 12d (3.6 g, 90%) as a yellow solid. MS, m/z 692 (M +
1)+.
4’,5’-Didehydro-5’-deoxy-2’-C-methyl -N2-(methoxytrityl)guanosine (13a). To the solution of
iodide 12a (11.5 g, 16.9 mmol) in dry THF (120 mL) was added DBU (12.87 g, 84.7 mmol), and
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heated overnight at 60 oC. The reaction was quenched with saturated NH4Cl solution. Usual work-
up and chromatography (MeOH in DCM from 1% to 5%) yielded compound 13a (5.5 g, 60%) as
a white solid. MS, m/z 552.3 (M + 1)+. 1H NMR (CD3OD): δ 7.47 (s, 1H), 7.22-7.34 (m, 12H),
6.83-6.85 (m, 2H), 5.39 (s, 1H), 4.47 (s, 1H), 4.46 (s,1H), 4.24 (s,1H), 3.76 (s, 3H), 0.63 (s, 3H).
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4’,5’-Didehydro-5’-deoxy-2’-C-ethynyl -N2-(methoxytrityl)guanosine (13b). Compound 13b
(2.0 g, 62%) as a white solid was synthesized from 4.0 g of 12b in the same manner as 13a. MS,
m/z 602.2 (M + 1)+. 1H NMR (CD3OD): δ 7.65 (s, 1H), 7.18-7.34 (m, 12H), 6.82-6.86 (m, 2H),
5.52 (s, 1H), 4.70 (t, J = 1.8 Hz, 1H), 4.46 (t, J = 2.2 Hz, 1H), 4.27 (t, J = 1.8 Hz, 1H), 3.76 (s,
3H), 2.80 (s, 1H).
4’,5’-Didehydro-2’,5’-dideoxy-2’-fluoro-2’-C-methyl-N2-(methoxytrityl)guanosine
(13c).
Compound 13c (1.9 g, 67%) was obtained from 12c using the same procedure as for the compound
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13a. MS, m/z 554.4 (M + 1)+. H-NMR (CDCl3): δ 7.12-7.32 (m, 13H), 6.71-6.73 (m, 2H), 5.56
(d, J = 14.8 Hz, 1H), 4.40-4.62 (m, 3H), 3.71 (s, 3H), 0.75 (d, J = 22.4 Hz, 3H).
4’,5’-Didehydro-2’,5’-dideoxy-2’-fluoro-2’-C-ethynyl-N2-(methoxytrityl)guanosine
(13d).
Compound 13d (3.0 g, 83%) was obtained as a yellow solid from 4.4 g of 12d using the same
procedure as for the compound 13a. MS, m/z 564 (M + 1)+.
2’5’-Dideoxy-2’,4’-difluoro-5’-iodo-2’-C-methyl-3’-O-benzoyl-N2-(methoxytrityl)guanosine
(15c). To the ice-cold solution of 13c (3.0 g, 5.4 mmol) in CH3CN (20 mL) was added Et3N.3HF
(0.65 g, 4.1 mmol) followed by NIS (1.53 g, 6.78 mmol). The reaction mixture was kept at rt for
2 h. After usual work-up C-4’ stereoisomers were separated by RP-HPLC (Waters X-bridge
column 150 × 25 mm, 5 µm; gradient of 0.1% NH4CO3 in water and in CH3CN from 0% to 70%).
To the solution of desired R-isomer 14c (0.6 g, 0.9 mmol) in pyridine (10 mL) was added BzCl
(145 mg, 1 mmol) and kept at rt for 3 h. After usual work-up and chromatography (25% to 50%
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EtOAc in hexane) was compound 15c (0.55 g, 13% for two steps) isolated as a white solid. H-
NMR (CDCl3):  8.08 (s, 1H), 7.10-7.97 (m, 17H), 6.70-6.72 (m, 2H), 5.86 (d, J = 17.2 Hz, 1H),
3.49-3.68 (m, 6H), 0.84 (d, J = 22.4 Hz, 3H).
4’-Fluoro-2’,3’,5’-tri-O-benzoyl-2’-C-methyl-N2-(methoxytrityl)guanosine (16a). To an ice-
cold solution of 13a (2 g, 3.6 mmol) in DCM (80 mL) was added AgF (2.5 g, 20 mmol) and
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solution of I2 (2 g, 8 mmol) in DCM (80 mL). The reaction mixture was stirred at rt for 3 h.
Reaction mixture was quenched with solution of Na2S2O3 and NaHCO3. After usual work-up the
crude 4’-fluorinated nucleoside 14a (1.7 g) was dissolved in DCM (250 mL). DMAP (1.1 g, 9.6
mmol) was added, followed by Et3N (1.4 mL, 9.6 mmol) and BzCl (0.8 mL, 7.2 mmol). The
reaction mixture was kept at rt overnight. After usual work-up and chromatography (3% MeOH in
DCM) benzoate 15a (1.5 g, 2 mmol) was isolated. It was dissolved in HMPA (40 mL), NaOBz
(7.2 g, 20 mmol) and 15-crown-5 (4.1 g, 20 mmol) were added and the mixture was stirred for 48
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h at 60 C. Usual work-up and chromatography (50% EtOAc in hexane) yielded 480 mg of 16a
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(15% yield for 3 steps). MS, m/z 900.4 (M + 1)+. H-NMR (CD3OD): δ 7.81-7.95 (m, 7H), 7.60-
7.66 (m, 8H), 7.39-7.51 (m, 2H), 7.19-7.31 (m, 13H), 6.36 (s, 1H), 5.95 (d, J = 18 Hz, 1H), 4.50-
4.70 (m, 2H), 3.72 (s, 3H), 1.41 (s, 3H).
4’-Fluoro-2’,3’,5’-tri-O-benzoyl-2’-C-ethynyl-N2-(methoxytrityl)guanosine (16b). Compound
16b (180 mg) was synthesized in 6% yield (for 3 steps) from 13b by the sequence of
transformations described above for 16a. MS, m/z 910.4 (M + 1)+. 1H NMR (CD3OD): δ 7.83-7.93
(m, 7H), 7.19-7.83 (m, 21H), 6.76-6.78 (m, 2H), 6.36 (s, 1H), 5.92-5.97 (m, 1H), 4.50-4.70 (m,2H),
3.72 (s, 3H), 1.40 (s, 3H).
2’-Deoxy-2’,4’-difluoro-2’,3’,5’-tri-O-benzoyl-2’-C-ethynyl-N2-(methoxytrityl)guanosine
(16d). Compound 16d was synthesized from 13d by the sequence of transformations described for
16a. The yield was 430 mg, 11% for 3 steps. MS, m/z 808 (M + H)+. 1H-NMR (CDCl3):  6.80-
7.78 (m, 27H), 6.22 (d, J = 14.8 Hz, 1H), 4.30 (t, J = 9.2 Hz, 1H), 3.70 (s, 3H), 2.42 (d, J = 5.6
Hz, 1H).
4’-Fluoro-2’-C-methyl-N2-(methoxytrityl)guanosine (17a). The mixture of protected
nucleoside 16a (400 mg, 0.44 mmol) and n-butylamine (1.5 mL) was kept overnight at rt and then
evaporated. The residue was purified by chromatography (MeOH in DCM from 4% to 15%) to
yield 206 mg (80%) of the target 17a. MS, m/z 586 (M+1)+. 1H-NMR (CD3OD):  8.01(s, 1H),
7.24-7.34 (m, 12H) 6.84-6.86 (m, 2H), 5.54 (s, 1H), 4.09 (d, J = 24.0 Hz, 1H), 3.76 (s, 3H), 3.64-
3.73 (m, 2H), 0.59 (s, 3H). 19F-NMR (CD3OD)  -123.24.
4’-Fluoro-2’-C-ethynyl-N2-(methoxytrityl)guanosine (17b). Compound 17b (100 mg, 86%)
was obtained from 16b using the same procedure as for the compound 17a. MS, m/z 598 (M +
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1)+. H-NMR (CD3OD):  7.89 (s, 1H), 7.18-7.35 (m, 12H), 6.83-6.87 (m, 2H), 5.64 (s, 1H),
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4.45-4.58 (m, 1H), 3.76 (s, 3H), 3.62-3.70 (m, 2H), 2.69 (s, 1H).
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2’-Deoxy-2’,4’-difluoro-2’-C-methyl-N2-(methoxytrityl)guanosine (17c). Solution of 15c (0.65
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g, 0.8 mmol), NaOBz (1.15 g, 8 mmol), and 15-crown-5 (1.77 g, 8 mmol) in DMF (10 mL) was
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stirred for 48 h at 100 C. Usual work-up and chromatography (15% to 50% EtOAc in hexane)
yielded 500 mg of 16c. Obtained nucleoside was treated with NH3/MeOH (50 mL) for 18 h at rt.
Solvent was evaporated and the residue was purified by chromatography (MeOH in DCM from
1% to 4%) to give 17c as a white solid. Yield: 231 mg (49% for 2 steps). MS, m/z 590.3 (M + 1)+.
1H-NMR (CD3OD):  7.93 (s, 1H), 7.22-7.31 (m, 12H), 6.83-6.85 (m, 2H), 5.65 (d, J = 16.0 Hz,
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1H), 4.21-4.30 (m, 1H), 3.71-3.77 (m, 5H), 0.75 (d, J = 24.0 Hz, 3H). F-NMR (CD3OD):  -
125.1, -162.4.
4’-Fluoro-2’-C-methylguanosine (4). A solution of 17a (261 mg, 0.44 mmol) in CH3CN (0.5 mL)
and 4N HCl/dioxane (0.05 mL) was kept at rt for 45 min and then evaporated. The crude residue
was treated with CH3CN, precipitate was filtered, washed with CH3CN, and dried to yield 4 (112
mg, 80%). MS, m/z 314.6 (M -1)-. 1H-NMR (CD3OD):  9.00 (s, 1H), 6.23 (s, 1H), 4.44 (d, J =
19.5 Hz, 1H), 3.75-3.81 (m, 2H), 1.05 (s, 3H). 19F-NMR (CD3OD):  -123.40.
4’-Fluoro-2’-C-ethynylylguanosine (5). Compound 5 was synthesized as described for 4 starting
from 17b. Yield: 104 mg (73%). MS, m/z 326.6 (M + 1)+ 1H-NMR (CD3OD):  7.96 (s, 1H), 6.29
.
(s, 1H), 4.75 (d, J = 18.4 Hz, 1H), 3.79 (m, 2H), 2.80 (s, 1H). 19F (DMSO-d6):  -124.24.
2’-Deoxy-2’,4’-difluoro-2’-C-methylguanosine (6). Compound 6 was synthesized as described
for 4 starting from 17c. Yield: 70 mg (81%). MS, m/z 317.6 (M-1)-. 1H-NMR (DMSO-d6):  11.08
(s, 1H), 8.16 (s, 1H), 6.87 (br, 2H), 6.28 (d, J = 1.6 Hz, 1H), 4.41 (d, J = 2.2 Hz, 1H), 3.70-3.63
(m, 2H), 1.05 (d, J = 2.3 Hz, 3H). 19F-NMR (DMSO-d6):  -122.0, -158.1; 13C (DMSO-d6): δ
156.6, 154.0. 150.7, 134.3, 116.6, 116.6 (d, J = 233 Hz), 98.6(d, J = 184 Hz), 88.8 (d, J = 43 Hz),
71.3, 59.4 (d, J = 40 Hz), 17.2 (d, J = 25 Hz).
2’-Deoxy-2’,4’-difluoro-2’-C-ethynylguanosine (7). The mixture of protected nucleoside 16d
(430 mg, 0.47 mmol) and n-butylamine (1.5 mL) was kept overnight at rt and then evaporated
yielding 17d. The solution of crude 17d in CH3CN (0.5 mL) and 4N HCl/dioxane (0.05 mL) was
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kept at rt for 45 min and then evaporated. The crude residue was treated with CH3CN, precipitate
was filtered, washed with CH3CN, and dried to yield 7 (60 mg, 36%). MS, m/z 326.5 (M -1)-. 1H-
NMR (DMSO-d6): δ 10.75 (s, 1H), 7.79 (s, 1H), 6.60 (s, 2H), 6.36 (d, J = 8.0 Hz, 1H), 5.71 (br,
1H), 4.8 (br, 1H), 3.69-3.62 (m, 2H), 3.00-2.70 (m, 1H). 19F-NMR (DMSO-d6):  -122.0, -158.1.
13C (DMSO-d6): δ 156.5, 153.9, 150.9, 134.2, 116.4, 116.3 (d, J = 235 Hz), 93.2 (d, J = 186 Hz),
88.1 (d, J = 42 Hz), 82.7 (d, J = 8 Hz), 75.2 (d, J = 31 Hz), 73.5 (d, J = 20 Hz), 59.4 (d, J = 39
Hz).
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9-(2- O-Benzoyl-5-O-benzyl-3-deoxy-3-fluoro-β-D-ribofuranosyl)-6-chloro-2-aminopurine
(19). To a solution of 6-chloro-9H-purin-2-amine (9.4 g, 55.4 mmol) in CH3CN (100 mL) was
added N,O-bis(trimethylsilyl)acetamide (33.8 g, 166.2 mmol). The resulting solution was stirred
for 2 h at 80 oC. To this reaction mixture was added a solution of compound 18 (10 g, 27.8
mmol) in CH3CN (100 mL), and trimethylsilyl trifluoromethanesulfonate (25 g, 112.6 mmol) at
0 oC. The resulting solution was let to react overnight at 80 oC. Usual work-up and
chromatography (50% EtOAc in hexanes) yielded nucleoside 19 as a white solid (10 g, 72%).
MS, m/z 498 (M+1)+. 1H-NMR (CDCl3): δ 8.2-8.50 (m, 3H), 7.54-7.61 (m, 1H), 7.33-7.51 (m,
7H), 6.46 (d, J = 7.6 Hz, 1H), 5.96-6.04 (m, 1H), 5.58-5.59 (m, 1H), 5.36-5.46 (m, 2H), 4.57-
4.71 (m, 3H), 3.71-3.84 (m, 2H). 19F-NMR (CDCl3): δ -197.52.
9-(5-O-Benzyl-3-deoxy-3-fluoro-β-D-ribofuranosyl)-6-chloro-2-(tritylamino)purine (20). To
a solution of nucleoside 19 (3 g, 6 mmol) in DMF (50 mL) was added triethylamine (3.4 mL, 24.0
mmol), DMAP (298 mg, 2.4 mmol), and chlorotriphenylmethane (5.5 g, 20 mmol). The resulting
solution was stirred for 24 h at 45 oC. The reaction was then quenched with MeOH (20 mL) and
concentrated under vacuum. The residue was purified by column chromatography (35% EtOAc in
hexanes) to get 3.6 g (81%) of N-tritylated nucleoside as a white solid. MS, m/z 740 (M+1)+.
Obtained nucleoside (3.0 g, 3.9 mmol) in methanolic ammonia (60 mL) was stirred for 4 h at rt.
Evaporated residue was purified by column chromatography (50% EtOAc in hexanes) to yield
compound 20 (2.0 g 78%) as a white solid MS, m/z 636.3 (M+1)+.
9-(5-O-Benzyl-3-deoxy-3-fluoro-2-oxo-β-D-ribofuranosyl)-6-chloro-2-(tritylamino)purine
(21). To a solution of compound 20 (2.0 g, 3.0 mmol) in DCM (20 mL) was added iodobenzene
diacetate (1.3 g, 4.0 mmol) and acetic acid (2 mL). It was followed by dropwise addition of a
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solution of TEMPO (0.15 g, 0.9 mmol) in DCM (5 mL) at 0 oC. The resulting solution was stirred
for 16 h at rt then diluted with DCM (40 mL) and quenched with solution of Na2S2O3. The organic
layer was washed with brine, dried (Na2SO4) and concentrated. The crude product was crystallized
from DCM /heptane (1:1 v/v) to yield ketone 21 (2.0 g, 100%) as a red solid. MS, m/z 652.9
(M+1+H2O)+ .
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9-(5-O-Benzyl-2,3-dideoxy-2,3-difluoro-2-C-methyl-β-D-ribofuranosyl)-6-chloro-2-
aminopurine (22). To a solution of methylmagnesium bromide (3M in ethyl ether, 10.5 mL) was
added a solution of compound 21 (1.0 g, 1.6 mmol) in THF (7 mL) dropwise with stirring at -78
oC. The resulting solution was stirred for 2 h at -30 oC, and then quenched with sat. aq. NH4Cl (20
mL). Usual work-up and column chromatography purification (50% EtOAc in hexanes) yielded
206 mg (20%) of 2’-methylated nucleoside as a yellow oil. MS, m/z 650 (M+1)+. DAST (102 mg,
0.63 mmol) was added dropwise to the solution of obtained nucleoside in DCM (10 mL) at -78oC.
The resulting solution was stirred for 2 h at rt, then quenched with aq. NaHCO3. Usual work-up
and column chromatography purification (25% EtOAc in hexanes) yielded 2’-fluorinated
nucleoside (45 mg, 22%) as a white solid. MS, m/z 652 (M+1)+. A mixture of obtained nucleoside
in MeOH (0.34 mL) was treated with AcOH (0.30 mL) for 12 h at 50 oC. Solvent was concentrated,
and the residue purified by silica gel chromatography (50% EtOAc in hexanes) to yield compound
1
22 (29 mg, 98%) as a yellow oil. MS, m/z 410 (M+1)+. H-NMR (CDCl3): δ 8.32 (s, 1H), 7.28-
7.41 (m, 5H), 6.20 (d, J = 16.8 Hz, 1H), 5.52 (s, 2H), 5.15-5.46 (m, 1H), 4.63-4.71 (m, 2H), 4.43-
19
4.49 (m, 1H), 3.96-3.99 (m, 1H), 3.76-3.82 (m, 1H), 1.22-1.34 (m, 3H). F-NMR (CDCl3): δ -
163.92, -221.81.
2’, 3’-Dideoxy-2’,3’-difluoro-2’-C-methylguanosine (9). To a solution of compound 22 (25 mg,
0.06 mmol) in MeOH (2 mL) was added 2-mercaptoethanol (23.8 mg, 0.30 mmol) and sodium
o
methylate (16.5 mg, 0.31 mmol). The resulting solution was stirred for 12 h at 65 C. The pH
value was adjusted to 7 with AcOH and the solution was concentrated. Residue was purified by
silica gel chromatography (10% MeOH in DCM) to yield 5-benzylated guanosine analogue (29
mg) as a white solid. MS, m/z 392 (M+H)+. A solution of obtained compound in DCM (0.5 mL)
was cooled down to -78 oC. BCl3 (1 M in heptane, 1 mL) was added and the reaction mixture was
left for 0.5 h at the same temperature. Reaction was quenched with MeOH and the mixture
concentrated. The crude product was purified by Prep-HPLC (XBridge BEH Phenyl OBD Prep
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column, 19 × 150 mm; using 10-25% CH3CN in 10 mmol aq. NH4HCO3 for elution.
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Difluoronucleoside 9 (10 mg, 55%) was isolated as a white solid. MS, m/z 302.1 (M+1)+. H-
NMR (CDCl3): δ 8.06 (s, 1H), 6.12-6.18 (m, 1H), 5.33-5.53 (m, 1H), 4.30 (m, 1H), 4.01-4.05 (m,
7
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1H), 3.87-3.91 (m, 1H), 1.20 (d, J = 22.4 Hz, 3H), F-NMR (CDCl3): δ -163.03, -222.52. C
(DMSO-d6):        dd, J = 183 Hz, 13 Hz), 89.9 (dd, J = 194
Hz, 15 Hz), 87.3 (dd, J = 38 Hz, 4 Hz), 59.6, 16.4 (d, J = 25 Hz).
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9-(5-O-Benzyl-2,3-dideoxy-3-fluoro-2-C-methylene-β-D-ribofuranosyl)-6-chloro-2-
(acetylamino)purine (23). To a solution of methyltriphenylphosphonium bromide (28.2 g, 78.3
mmol) in THF (400 mL) under inert atmosphere was added lithium bis(trimethylsilyl)amide (79
mL, 1 M in THF) dropwise with stirring at 0 oC. The resulting solution was stirred for 2 h at rt and
then cooled to 0 oC. THF solution of ketone 21 (20 g, 31.5 mmol) was added. The resulting mixture
was left for 4 h at rt. Reaction was quenched with sat. aq. NH4Cl. Usual work-up and column
chromatography purification (30% EtOAc in hexanes) yielded 2’-methylene derivative (6.8 g, 34%)
as a yellow solid. MS, m/z 632 (M+H)+. Methanolic solution of this nucleoside was treated with
AcOH (51 ml) for 16 h at 50 oC. Solvents were evaporated and detritylated nucleoside was purified
by silica gel chromatography (30% EtOAc in hexanes) to get 2.5 g (60%) of white solid. Obtained
nucleoside was dissolved in pyridine (50 mL) and DCM (100 mL). AcCl (1.45 g, 18.5 mmol) was
added dropwise and the reaction mixture was kept at rt for 1 h. Reaction was quenched with MeOH
and solvents were evaporated. Product was purified by silica gel chromatography (50% EtOAcin
hexanes) to yield nucleoside 23 (1.8 g, 65%) as a white solid. MS, m/z 432 (M+1)+.
9-(5-O-Benzyl-2-chloro-2,3-dideoxy-3-fluoro-2-C-methyl-β-D-ribofuranosyl)-6-chloro-2-
(acetylamino)purine (25). To a solution of compound 23 (570 mg, 1.32 mmol) in dioxane (15
mL) under inert atmosphere was added reagent 24 (24 mg, 0.04 mmol) and 4-toluene sulfonyl
o
chloride (5.0 g, 26.23 mmol). The resulting solution was stirred for 1.5 h at 30 C followed by
dropwise addition of a solution of phenylsilane (143 mg, 1.32 mmol) in EtOH (10 mL). The
resulting solution was kept for an additional 1 h at 30 oC. Solvents were evaporated and product
was purified by silica gel chromatography (25% EtOAc in hexanes) to get nucleoside 25 (180 mg,
29%) as a yellow solid. MS, m/z 468.1 (M+1)+.
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9-(2,3-Dideoxy-2-chloro-3-fluoro-2-C-methyl-β-D-ribofuranosyl)-6-chloro-2-
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(acetylamino)purine (26). To a solution of compound 25 (60 mg, 0.13 mmol) in DCM (1.5 mL)
under inert atmosphere was added borontrichloride (1.2 mL, 1 M in hexane) dropwise at -78 oC.
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9
o
o
The resulting solution was stirred for 2 h at -78 C and 3 h at -30 C. The reaction was then
quenched by the addition of MeOH/DCM (1.5 mL, 1/1). The resulting mixture was concentrated
under reduced pressure to dryness to yield 45 mg of compound 26 which was used without
purification for further reaction. MS, m/z 378.2 (M+1)+.
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2’,3’-Dideoxy-2’-chloro-3’-fluoro-2’-C-methylguanosine (10). To a solution of nucleoside 26
(200 mg, 0.53 mmol) in MeOH (20 mL) was added 2-mercaptoethanol (206 mg, 2.64 mmol) and
o
NaOMe (476 mg, 30% in MeOH). The resulting solution was stirred for 3 h at 60 C. The pH
value of the solution was adjusted to 7 with acetic acid. The resulting mixture was concentrated
and purified by Prep-HPLC (SunFire C18 OBD Prep Column, 19 × 100 mm; 15-35% MeCN in
water). Guanosine 10 (44 mg, 26%) was isolated as a white solid. MS, m/z 318.1 (M+1)+. H-
1
NMR (CD3OD): δ 8.16 (s, 1H), 6.33 (s, 1H), 5.46-5.25 (m, 1H), 4.28-4.37 (m, 1H), 3.94-4.03 (m,
1H), 3.78-3.88 (m, 1H), 1.40 (s, 3H). 19F-NMR (CD3OD): δ -208.26. 13C (DMSO-d6):
       d, J = 196 Hz), 89.8 (d, J = 4 Hz), 80.6 (d, J = 22 Hz),
73.9 (d, J = 14 Hz), 59.2, 22.3.
9-(2-Deoxy-2-fluoro-2-C-methyl-β-D-ribofuranosyl)-6-ethoxy-2-(methoxytritylamino)-
purine (28). To a solution of compound 27 (12.5 g, 23.8 mmol) in DCM (50 mL) were added
AgNO3 (8.1 g, 47.6 mmol), collidine (5.8 g, 47.6 mmol) and MMTrCl (11 g, 35.6 mmol). Reaction
mixture was kept at rt overnight, quenched with MeOH and filtered. Solvents were evaporated and
the residue purified by column chromatography (5% MeOH in DCM) to get 16 g (84%) of N-
tritylated nucleoside. A solution of this product in EtOH (150 mL) was treated with NaOEt (50
o
mL, 2N in ethanol) at 0 C. Resulting reaction mixture was kept at rt for 1 h. The pH was then
adjusted to 7 with conc. aq. NH4Cl. Usual work-up and column chromatography purification (5%
MeOH in DCM) afforded 10.9 g (98%) of nucleoside 28. MS, m/z 600.5 (M+1)+. H-NMR
1
(DMSO-d6): δ 8.13 (s, 1H), 7.51 (s, 1H), 7.15-7.30 (m 13H), 6.80 (m, 2H), 5.61 (d, J = 7.2 Hz,
1H), 5.25 (t, J = 5.0 Hz, 1H), 4.05 (br, 2H), 3.80 (m, 2H), 3.68 (s, 3H), 3.61 (m, 1H) 1.15 (br, 3H),
0.75 (br, 3H).
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(methoxytritylamino)purine (29). Nucleoside 28 (10 g, 16.5 mmol), triphenylphosphine (5.4 g,
20.6 mmol) and imidazole (1.6 g, 24.7 mmol) were suspended in THF (30 mL) and the resulting
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9
o
mixture cooled to 10 C. Solution of iodine (5 g, 20 mmol) in THF (20 mL) was added and the
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reaction mixture was stirred at rt for 5 h. The reaction was quenched with aq. Na2S2O3 Usual work-
up and purification by column chromatography (2% MeOH in DCM) yielded nucleoside 29 (8.9
g, 75%) as a yellow solid. MS, m/z 710.2 (M+H)+.
9-(4,5-Didehydro-2,5-dideoxy-2-fluoro-2-C-methyl-β-D-ribofuranosyl)-6-ethoxy-2-
(methoxytritylamino)purine (30). To a solution of compound 29 (8 g, 11.3 mmol) in THF (40
mL) was added DBU (2.5 mL, 17 mmol) and the mixture was heated at 70-75 ⁰C for 3 h. Reaction
was quenched with 1M solution of citric acid. Usual work-up and purification by column
chromatography (20% EtOH in hexane) yielded nucleoside 30 (2.4 g, 35%) as a white solid. MS,
m/z 582.4 (M+1)+. 1H-NMR (CDCl3): δ 7.42 (s, 1H), 7.19-7.29 (m 12H), 6.78 (m, 2H), 6.30 (s,
1H), 5.8 (br, 1H), 4.6, 4.5, 4.3 (3 br, 4H), 3.77 (m, 2H), 3.68 (s, 3H), 3.61 (m, 1H) 1.35 (br, 3H),
0.75 (br, 3H).
9-(2,5-Dideoxy-2,4-difluoro-5-iodo-2-C-methyl-β-D-ribofuranosyl)-6-ethoxy-2-
(methoxytritylamino)purine (31). To a stirred cold (0 oC) solution of nucleoside 30 (4.8 g, 8.2
mmol) in DCM (50 mL) was added triethylamine trihydrofluoride (2 mL, 12.6 mmol) followed by
addition of NIS (2.7 g, 12 mmol). Reaction was allowed to proceed for 2h at the same temperature
and quenched with saturated solutions of NaHCO3 and Na2S2O3 (1:1). Usual work-up and
purification by column chromatography (20% EtOH in hexane) afforded 3.5 g (60%) of nucleoside
31. MS, m/z 728.1 (M+1)+.
9-(3,5-Di-O-benzoyl-2-deoxy-2,4-difluoro-2-C-methyl-β-D-ribofuranosyl)-6-ethoxy-2-
(methoxytritylamino)purine (32). To a solution of nucleoside 31 (2.1 g, 2.9 mmol) in pyridine
(40 mL) was added BzCl (464 mg, 3.3 mmol) and the reaction mixture was kept at rt overnight.
Usual work-up and chromatography on silica gel (20% EtOAc in hexane) yielded 2g of 3’-O-
benzoylated product. To a solution of this compound in DMF (60 mL) were added NaOBz (3.45
g, 24 mmol) and 15-crown-5 (5.31 g, 24 mmol) and the reaction mixture was stirred at 100 oC for
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