Synthesis and Anti-HCV Activities of 4′-Fluoro-2′-Substituted Uridine Triphosphates and Nucleotide Prodrugs: Discovery of 4′-Fluoro-2′- C-methyluridine 5′-Phosphoramidate Prodrug (AL-335) for the Treatment of Hepatitis C Infection

Article abstract of DOI:10.1021/acs.jmedchem.9b00143

We report the synthesis and biological evaluation of a series of 4′-fluoro-2′-C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC₅₀ values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC₅₀ values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.

Full text of DOI:10.1021/acs.jmedchem.9b00143

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Article
Synthesis and Anti-HCV Activities of 4’-Fluoro-2’-Substituted
Uridine Triphosphates and Nucleotide Prodrugs: Discovery
of 4’-Fluoro-2’-C-Methyluridine 5’-Phosphoramidate
Prodrug (AL-335) for the Treatment of Hepatitis C Infection
Guangyi Wang, Natalia Dyatkina, Marija Prhavc, Caroline Williams, Vladimir
Serebryany, Yujian Hu, Yongfei Huang, Jinqiao Wan, Xiangyang Wu, Jerome Deval,
Amy Fung, Zhinan Jin, Hua Tan, Kenneth Shaw, Hyunsoon Kang, Qingling Zhang,
Yuen Tam, Antitsa Stoycheva, Andreas Jekle, David Smith, and Leonid Beigelman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00143 • Publication Date (Web): 05 Apr 2019
Downloaded from http://pubs.acs.org on April 6, 2019
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Synthesis and Anti-HCV Activities of 4’-Fluoro-2’-Substituted Uridine Triphosphates and
Nucleotide Prodrugs: Discovery of 4’-Fluoro-2’-C-Methyluridine 5’-Phosphoramidate
Prodrug (AL-335) for the Treatment of Hepatitis C Infection
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1*
1,a
1,a
Guangyi Wang, Natalia Dyatkina, Marija Prhavc, Caroline Williams, Vladimir Serebryany,
Yujian Hu, Yongfei Huang, Jinqiao Wan, Xiangyang Wu, Jerome Deval, Amy Fung,1
2
2
2,b
2
1,a
1
1,a
1
1
1,a
1
Zhinan Jin, Hua Tan, Kenneth Shaw, Hyunsoon Kang, Qingling Zhang, Yuen Tam, Antitsa
Stoycheva, 1,a Andreas Jekle, 1,a David B. Smith, 1,a Leonid Beigelman1,a
1
Janssen BioPharma, Inc., South San Francisco, California, 94080, USA
2
Department of Medicinal Chemistry, WuXi AppTec, Shanghai 200131, P.R. China
Abstract: We report synthesis and biological evaluation of a series of 4’-fluoro-2’-C-substituted
uridines. Triphosphates of the uridine analogues exhibited potent inhibition of HCV NS5B
polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the
phosphoramidate prodrugs of these uridine analogues demonstrated very potent activity with EC50
values as low as 20 nM. A lead compound (53) demonstrated high levels of the NTP in vitro in
primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose.
Compound 53 (AL-335) was selected for clinical development where it showed promising results
in Phase 1 and II trials.
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INTRODUCTION
Hepatitis C is a major liver disease caused by the hepatitis C virus (HCV). HCV can cause both
acute and chronic hepatitis, ranging in severity from a mild illness lasting a few weeks to a serious,
0
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1
lifelong illness. Globally, an estimated 71 million people have chronic hepatitis C infection. A
significant number of those who are chronically infected will develop cirrhosis or liver cancer.
Approximately 399,000 people die each year from hepatitis C, mostly from cirrhosis and
1
2
hepatocellular carcinoma. The standard of care for hepatitis C is changing rapidly. Sofosbuvir,
3
4
daclatasvir and sofosbuvir/ledipasvir combination are among the preferred regimens in the WHO
guidelines. Therapy with these direct antiviral agents can achieve cure rates above 95% after a
typical treatment period of 12 weeks. However, access to HCV treatment remains low though it is
improving. In 2015, of the 71 million persons living with HCV infection globally, only 1.1 million
1
of people were started on treatment. The long term use of these drugs may lead to viral resistance
and a higher rate of adverse effects over time. Therefore, a combination of safe and effective drugs
with a shorter treatment period is highly desirable. A shorter duration of treatment is expected to
increase treatment accessibility to patients infected with HCV.
Nucleoside analogues have a proven record as antiviral drugs as their phosphate metabolites may
selectively inhibit viral targets, particularly viral polymerases, and effectively prevent viral
5
replication. This class of antiviral drugs typically exhibits a high barrier to viral resistance, which
makes nucleotide analogues a “backbone” of direct acting antiviral combination therapies. Two
types of modified nucleosides emerged as inhibitors of HCV polymerase, including 2’-methyl-
6
and 4’-azido-nucleosides. The well-known HCV drug sofosbuvir, a 2’-methyluridine analogue,
2
is widely used in HCV therapies. However, development of tri-isobutyrate ester of 4’-
6
azidocytidine (Balaprivir) was discontinued due to safety reasons. Another nucleoside drug BMS-
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86094 (INX-08189), a 2’-C-methylguanosine prodrug, demonstrated very potent activity against
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a
7b
HCV infection. However, the drug failed in a Phase 2 clinical trial because of its severe toxicity.
Incorporation of 2’-C-methylguanosine into host RNA by human mitochondrial RNA polymerase
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5
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0
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0
HMRP) may have been at least partially responsible for the toxicity.8
(
In our research, we explored a variety of sugar modified nucleoside analogues to identify a potent
and safe nucleoside analogue for HCV infection. In this article, we report synthesis, HCV NS5B
inhibition, host polymerase inhibition, and HCV replicon activity of a series of uridine analogues
11
having 4’-fluoro-2’-C-substituted sugar moieties (Figure 1). For a lead prodrug (53), in vitro and
in vivo nucleoside triphosphate (NTP) formation is also presented. Compound 53 (AL-335) in
combination with simeprevir and odalasvir was evaluated in human Phase 1 and 2 clinical trials
and demonstrated promising efficacy and safety results,12a,b,13 achieving 100% sustained viral
response after 6-weeks of treatment in patients with genotype 1 HCV infection. Updated results
on clinical trials will be published elsewhere.
H
N
H
N
H
N
O
O
O
O
O
O
O
O
O
N
N
N
HO
HO
HO
R4
R2
R
F
R
HO
OH
HO
OH
HO
F
2
4
1a R = Me
2a R = Me
3a R = Me, R = H
3b R2 = Me, R4 = F
3c
1b R = FCH2
2b R = FCH2
2c R = ethynyl
2d R = vinyl
R2 = ethynyl, R4 = H
d R2 = ethynyl, R4 = F
1c R = ethynyl
3
2e R = Et
H
N
H
N
O
O
O
O
O
O
N
N
HO
HO
R
R
Cl
HO
Cl
HO
F
4
4
a R = H
b R = F
5b R = F
5c R = H
Figure 1. 2’-C-Substituted uridines and 4’-fluoro-2’-C-substituted uridines
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6
RESULTS AND DISCUSSION
Chemistry
Synthesis of compounds 2a, 2b and 2c is shown in Scheme 1. Previously we reported the synthesis
of these compounds,14a later it was repeated by Wang et al.14b Synthesis of 2b was started with
the known compound 6.15a The uracil base of 6 was protected with 4-methoxybenzyl and the sugar
hydroxyls with benzyl. The resulting 8 was converted to the 2’-hydroxymethyl derivative 9 in
good yield by oxidative cleavage and subsequent reduction. The fluorination of 9 was achieved by
mesylation and treatment with TBAF. Removal of PMB and debenzylation gave, in good yield,
the unprotected 11,15b which was subjected to iodination, followed by elimination, to give the
olefinic intermediate 13.
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0
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9
0
In general introduction of 4’-F was achieved by utilizing the strategy initially developed by Moffat
1
6
17
et al. in their synthesis of nucleocidine and later utilized by others. Iodofluorination of 13,
followed by benzoylation of the sugar hydroxyls yielded 16a in moderate yield. Substitution of
iodo with a benzoyloxy moiety to yield 17a was accomplished by heating 16a with sodium
benzoate. Compound 2b was obtained by debenzoylation of 17a with ammonia in good yield.
18
Compound 16b was prepared from the known 14, by the same sequence of reactions used for
1
6a. The iodo moiety of 16b was replaced with m-chlorobenzoyloxy by treatment with mCPBA
in a buffer (pH 4) to give 18 after debenzoylation. Compound 2a was prepared from known 1519
in a similar manner as 18. Compound 2c was prepared by converting 18 to the ketone 20, followed
by Grignard reaction and subsequent desilylation. Unlike the usual Grignard reaction where alkyl
is added to the α-face of the 2’-keto of a nucleoside, ethynyl was added primarily to the β-face to
give the desired 2c in good yield. NMR evidence (NOESY) for the configuration at C-2’ was not
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conclusive. However 2c readily formed 2’,3’-isopropylidene analogue which proved ribo
configuration.
Scheme 1.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
PMB
N
PMB
N
PMB
N
PMB
N
H
N
O
O
O
O
O
O
O
O
O
O
O
a
O
b
O
c
O
d
O
10
O
N
N
N
N
N
BzO
HO
BnO
BnO
BnO
OH
OBn
F
BzO
OBz
HO
OH
BnO
OBn
BnO
BnO
OBn
6
7
8
9
H
N
H
N
H
H
H
O
O
O
O
O
N
N
N
O
O
O
O
O
e
O
f
O
g
O
h or i
O
j or k
N
N
N
R1
OR2
N
R1
OR2
N
R1
OR2
4
HO
I
I
R O
F
F
3
F
F
3
3
HO
OH
HO
OH
R O
R O
R O
12
13 R1 = CH F, R2 = R3 = H
16a R1 = CH F, R2 = R3 = Bz
16b R1 = H, R2 = R3 = Bz
16c R1 = Me, R2 = R3 = Ac
17a R1 = CH F, R2 = R3 = R4 =Bz
11
2
2
2
4 R1 = H, R2 = R3 = H
17b R1 = H, R2 = R3 = Bz, R4 = H
17c R1 = Me, R2 = Ac, R3 = R = Bz
1
1
5 R1 = Me, R2 = R3 = Ac
4
H
N
H
N
H
H
O
O
O
O
O
O
N
O
N
O
O
l or m
n
o
O
O
N
p
O
N
N
O
N
O
O
HO
HO
R1
F
F
F
F
HO
OH
TIPDS
TIPDS
O
OH
O
HO
OH
2b R1 = CH2F
19
20
2c
1
2
8 R1 = H
a R1 = Me
Reagents and conditions: a) i. PMBCl, K
BnBr, NaH, DMF, rt, overnight, 46%; c) i. OsO
NaBH , rt, 30 min, 61% (3 steps); d) i. MsCl, DMAP, DCM, rt, 40 min, 90%; ii. TBAF, THF, 75 C, 3 d, 27%; e) i.
CAN, CH
THF/CH
2
CO
3
, DMF, rt, overnight; ii. NaOMe, MeOH, rt, 2.5 h, 75% (2 steps); b)
4
, NMMO, THF/H O, rt, 1 d; ii. NaIO , MeOH/H O/THF, rt, 2 h; iii.
2
4
2
o
4
o
3
CN/H
2
O, overnight,71%; ii. BCl
3
, -70 C, 2 h, 86%; f) PPh
3
, I
2
, pyridine, rt, overnight, 58%; g) DBU,
o
o
3
CN (2:1), 65 C, 2 h, 55%; h) i. TEA-3HF, NIS, CH
3
CN, rt, 30 min; ii. Bz
2
O, DMAP, pyridine, 50 C,
overnight, 35% for 16a, 59% (2 steps) for 16b; i) i. Ac
2
O, pyridine, rt, overnight; ii. AgF, I
2
, DCM, rt, 5 h, 40% (2
NOH, TFA, pH
/MeOH, rt, 5 h, 75% for 18, 25% for 2b; m) NH , rt,
CN, 80 C, 3 h, 80%; p) i.
o
steps) for 16c; j) NaOBz, 15-crown-5, DMF, 130 C, 6 h, 46% for 17a, 41% for 17c; k) 55% aq. Bu
4
4
, mCPBA, DCM, rt, overnight, 68% for 17b; l) 7 M NH
3
3
o
overnight, 84% for 2a; n) TIPDSCl
2
, pyridine, rt, overnight, 40%; o) IBX, CH
3
ethynylmagnesium bromide, THF, rt, 1 h; ii. TBAF, THF, rt, 30 min, 39% (2 steps).
Synthesis of 2d and 2e is shown is Scheme 2, starting from the known 1c.14 The 2’ and 3’-
hydroxyls of 1c were protected with cyclopentylidene and the 5’-hydroxyl was converted to the
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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4
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5
6
5
’-iodide. The resulting 22 was subjected to the acidic hydrolysis and elimination to yield the
olefin 23. Iodofluorination of 23, followed by benzoylation of the sugar, yielded the 4’-fluoro
intermediate 24. After hydroxydeiodination, the resulting 25 was converted to 2d by controlled
hydrogenation and subsequent hydrolysis. Similarly, 25 was converted to 2e.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2.
H
N
H
N
H
N
H
O
N
O
O
O
O
O
O
O
a
O
b
O
c
O
d
O
N
N
N
N
HO
HO
I
HO
OH
O
O
O
O
HO
OH
23
1c
21
22
Bz
Bz
Bz
N
H
N
O
N
O
O
O
O
O
O
N
O
e
f
g
O
N
O
N
O
N
O
N
HO
HO
HO
I
F
F
F
HO
F
BzO
BzO
OBz
O
BzO
OBz
OH
OBz
24
25
26
2d
h
Bz
N
H
N
O
O
O
O
N
O
N
i
HO
HO
F
F
HO
BzO
OBz
OH
27
2e
o
Reagents and conditions: a) TsOH, 1,1-dimethoxycyclopentane, DCE, 50 C, overnight, 70%; b)
i. TsCl, pyridine, rt, overnight; ii. NaI, acetone, reflux, overnight, 57% (2 steps); c) i. 80% AcOH,
o
o
6
3 2
0 C, 2 h, 79%; ii. DBU, THF, 65 C, 48%; d) i. TEA-3HF, NIS, CH CN, rt; ii. Bz O, DMAP,
o
pyridine, 50 C, 12% (2 steps); e) aq. TBAOH, , TFA, pH 4, mCPBA, rt, 24%; f) Lindlar catalyst,
3 2
H2 (40 psi), EtOAc, hexanes, rt, 1.5 h; g) 7 M, NH in MeOH, rt, 35% (2 steps); h) Pd/C, H , MeOH,
rt, 1.5 h, i) 7 M NH
3
in MeOH, rt, 21% (2 steps).
Synthesis of 4a20,30 started with 28 , which was converted with tetrabutylammonium chloride at
elevated temperature to the corresponding chloride via SN2 mechanism. Acidic hydrolysis
followed by benzoylation afforded 30 in good yield. Conversion of 30 to 31 was achieved by
21
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reduction and subsequent acetylation. Condensation of anomeric mixture of 31 with silylated
cytosine under Vorbrueggen condition gave the cytidine 32. Only β-cytidine was isolated and was
converted to the uridine 4a by heating with acetic acid, followed by debenzoylation and was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
identical to the reported 2’-chloro-2’-methyluridine.20
Scheme 3.
O
O
O
O
O
O
OAc
a
COOEt
Cl
c
d
b
O
BzO
BzO
COOEt
HO
BzO
Cl
BzO
Cl
O
O
O
S
29
30
31
O
28
H
N
H
N
O
O
N
O
N
O
NHBz
O
f
e
O
O
O
N
N
BzO
HO
BzO
BzO
Cl
HO
Cl
BzO
Cl
33
4a
32
Reagents and conditions: a) i. Bu
4
NCl, dioxane/H
2
O, reflux, overnight; ii. (MeO)
2
CMe
2
, HCl,
AlH,
rt, 3 h, b) i. HCl, EtOH, rt, 48 h; ii. BzCl, pyridine, rt, 45 min, 49% (4 steps); c) i. Li(t-Bu)
3
o
o
THF, -60 C, 3 h; ii. Ac
2
O, DMAP, THF, 0-5 C, 1 h, 79% (2 steps); d) i. N-Bz-cytosine,
o
(NH
4
)
2
SO
4
, HMDS, reflux, 4 h; ii. SnCl
4
, 65 C, PhCl, overnight, 45%; e) AcOH/H
2
O (9:1),
o
1
3
10 C, overnight, 89%; f. 10 M NH in MeOH, rt, overnight, 63%.
22
Reported synthesis of 5a via Vorbrueggen condensation using 1-O-acetyl sugar, 1-O-mesyl
sugar, or 35 under various conditions yielded predominantly the α-nucleoside with low yield of
desired β-isomer. In search of new synthetic route for 5a, we found that the 1-amino sugar 37 was
relatively stable, which could be used to build the uracil base on the sugar. In this way, α/β-mixture
2
3
(
roughly 1:1) of 5a was prepared from known 34, as shown in Scheme 4.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4.
O
O
OH
Cl
Br
b
TIPSO
TIPSO
TIPSO
TIPSO
a
Cl
F
F
F
34
35
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
N3
Cl
NH2
Cl
TIPSO
TIPSO
c
TIPSO
TIPSO
d
F
36
37
EtO
H
N
O
O
O
H
N
O
e
O
N
NH
TIPSO
TIPSO
HO
Cl
Cl
F
O
HO
F
5
a -mixture
c -isomer
3
8
5
Reagents and conditions: a) CBr
4
3
, Ph P, DCM, rt, 1 h,
o
9
2%; b) NaN
3
, DMF, 50 C, overnight, 89%; c) 20%
Pd(OH) /C, H
2
2
, EtOAc, rt, 2 h, 98%; d) (E)-3-
ethoxyacryloyl isocyanate, toluene, DCM, rt, 2 h, 94%,
o
e) i. HCl, EtOH/H
2
O, 85 C, 10 h; ii. NH
4
F, MeOH, 70
oC, 5 h, 74% (2 steps).
2
4
25
Compounds 3b, 3d, 4b and 5b were synthesized from 3a, 3c, 4a, and 5a, respectively, by
procedures similar to those described in Scheme 1. Reaction conditions are shown in the Scheme
5
.
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8
9
Scheme 5.
H
H
O
N
O
O
N
O
a
b
O
N
O
N
HO
I
R2
R2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R2'
R2'
2
HO
HO
2
2'
2'
3
a R = Me, R = F
40a R = Me, R = F
2
2'
2
2'
3
c R = ethynyl, R = F
40b R = ethynyl, R = F
2
2'
2
2'
4a R = Me, R = Cl
40c R = Me, R = Cl
2
2'
2
2'
5a R = Cl, R = F (-mixture)
40d R = Cl, R = F
H
H
O
N
O
O
N
O
c
d
O
N
O
N
I
R2
F
R2
R2'
HO
R2'
BzO
41a-d
42a-d
H
H
O
N
O
O
N
O
e
O
O
N
N
HO
HO
F
R2
R2'
F
R2
R2'
BzO
HO
2
2'
3
b R = Me, R = F
43a-d
2
2'
3d R = ethynyl, R = F
2
2'
4b R = Me, R = Cl
2
2'
5
b R = Cl, R = F
3 2
Reagents and conditions: a) PPh , I , pyridine, THF, rt,
overnight, 51% for 40a, 77% for 40b, 85% for 40c, 27%
o
for 40d; b) DBU, THF, 50 C, overnight, 60% for 41a,
6
9% for 41b, 45% for 41c, 78% for 41d; c) i. TEA-3HF,
NIS, CH CN, rt, 7 h; ii. BzCl, pyridine, rt, 5 h, 76% (2
steps) for 42a, 37% for 42b, 32% for 42c, 39% for 42d;
d) Bu NOH, TFA, pH 4, mCPBA, DCM, rt, 5 h, 55% for
3a, 78% for 43b, 41% for 43c, 57% for 43d; e) 7 M
3
4
4
3
NH in MeOH, rt, 5 h, 66% for 3b, 59% for 3d, 53% for
4
b, 81% for 5b.
All nucleosides in Figure 1 were converted to their 5’-triphosphates for testing in viral and human
polymerase assays. For testing in cell-based assays, most of the nucleosides in Figure 1 were
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
converted to their 5’-phosphoramidate prodrugs (44-52) as shown in Table 3. For the nucleoside
2
a, several 5’-phosphoramidate prodrugs (53-58) and 5’-bis-phosphoramidate prodrugs (59-60)
that have either a different alkyl ester of the alanine or a different substitutions on the phosphorus
were prepared, as shown in Table 4. Among them is a pair of P-isomeric prodrugs 53 and 54,
which were separated from 44 by HPLC. Chemical preparation of the triphosphates and the
prodrugs is described in the experimental section.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
X-Ray crystal structure of compound 53.
Absolute stereochemisty of compound 53 (AL-335) was assigned by X-ray crystallography with
resolution 0.83 Å. Confirmed chiralities of stereocenters are as follows: P1 is (S); C2 is (S); C3 is
(S); C4 is (R); C5 is (R); C18 is (S). Atom numbers of chiral centers are shown in Figure 2. Clearly,
compound 53 is the Sp isomer.
Figure 2. X-ray crystal structure of compound 53
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Inhibition of HCV NS5B polymerases
’-Fluoro-2’-C-substituted uridine triphosphates were tested in HCV NS5B polymerase assay,26
4
along with their 4’-H triphosphate analogues for comparison. As shown in Table 1, all NTPs except
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
for 2d-TP and 2e-TP are potent inhibitors of HCV NS5B with IC values ranging from 0.027 to
5
0
0
.86 M. Weak inhibition by 2d-TP and 2e-TP could be caused by the unfavorable shape of 2’-
C-vinyl and 2’-C-ethyl since 1c-TP, 2c-TP, 3c-TP and 3d-TP which have 2’-C-ethynyl are potent
inhibitors. It is noticible that 1c-TP and 2c-TP which have 2’-OH are approximately 10 fold more
potent than the corresponding 3c-TP and 3d-TP which have 2’-F. 1b-TP and 2b-TP which have
2
’-C-fluoromethyl are equipotent as 1a-TP and 2a-TP which have 2’-C-methyl, implicating that
the electron withdrawing nature of the fluorine did not exert a significant effect on the inhibition.
Compound 4a-TP which has 2’-α-chloro-2’-C-methyl exhibited very potent inhibition with an
IC value of 0.062 M, four fold more active than its closely related analogue 3a-TP, although
50
Cl has a larger size than F. 5c-TP containing 2’-β-Cl-2’-α-F has an IC value of 0.30 M,
5
0
equipotent to 3a-TP. Replacement of methyl with chlorine at the 2’ position appears well tolerated
although their inductive properties and electron densities are quite different. When fluorine is
introduced at 4’ position of the sugar, 2a-TP, 3b-TP, 3d-TP, 4b-TP and 5b-TP have basically
equipotent inhbitory effets on NS5B as the corresponding 4’-H NTPs, 1a-TP, 3a-TP, 3c-TP, 4a-
TP and 5c-TP. The 4’-F NTPs such as 2a-TP and 4b-TP showed very desirable inhibtion of NS5B
with IC values of 0.14 and 0.11 M, respectively. A preliminary conclusion is that introduction
5
0
of fluorine at the 4’ position does not alter inhibitory properties of NS5B dramatically and can lead
to more potent NTPs.
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Inhibition of HCV polymerase NS5B by
uridine triphosphatesa,b
H
N
O
O
O
N
HO
R4
R2
R2'
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HO
NTP
R2
Me
FCH
R2’
R4
IC50, M
1a-TP30
1b-TP
1c-TP
2a-TP
OH
OH
OH
OH
OH
OH
OH
OH
F
H
H
H
F
0.26
0.28
0.027
0.14
0.15
0.039
6.4
2
-
ethynyl
Me
2
b-TP
c-TP
d-TP
FCH
2
-
F
2
ethynyl
vinyl
Et
F
2
F
2
e-TP
F
84
3
a-TP30
Me
H
F
0.27
0.86
0.33
0.34
0.062
0.11
0.19
0.30
3
b-TP
Me
F
3
c-TP
ethynyl
ethynyl
Me
F
H
F
3
d-TP
F
4
a-TP30
Cl
H
F
4
b-TP
b-TP
Me
Cl
5
Cl
F
F
5
c-TP
Cl
F
H
a
b
IC50 indicates a concentration at which the activity of
HCV polymerase NS5B is inhibited by 50%.
Each IC50 value is an average of n ≥ 2 determinations.
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9
As shown in Table 1, several 4’-F NTPs are highly inhibitory to HCV NS5B polymerase. To
2
7
unravel their mechanism of action, 2a-TP was tested in a chain termination assay. As shown in
Figure 3, the dimer GG in the lane 2 and 3 could elongate to the trimer GGC in the presence of
CTP or CTP and ATP. After adding UTP, the GG dimer could elongate to a 19-mer (lane 4).
However, the elongation stopped after 2a-TP, as a replacement of UTP, was incorporated into the
sequence (lane 5). A similar termination was observed when UTP was replaced by 3’-dUTP. The
results indicate that 2a on the elongating sequence acts as an effective chain teminator.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
9-mer -
Primer
5’-*GG-3'
Template 3’- CCG AUG UUA UUA AUA A-5'
Lane
NTP added
Product sequence
1
2
3
4
5
6
H2O
C
5'- GG
5'- GGC
5'- GGC
C,A
4-mer -
3-mer -
C,A,U
C,A,2a
5'- GGC UAC AAU AAU UAU ACA U
5'- GGC 2a
2
-mer -
C,A,3’-dU 5'- GGC 3’dU
1
2 3 4 5 6
Figure 3. Chain termination of HCV polymerase NS5B mediated RNA Synthesis by 2a-TP.
is
Inhibition of human polymerases
To assess the selectivity between viral and human polymerase inhibitions, selective NTPs
from Table 1 were tested for inhibition of human DNA polymerases α, β, and  as well as human
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
10
RNA polymerase II. The results are listed in Table 2. The known 2’-fluoro-2’-C-methyl-UTP
(
3a-TP) and 5c-TP as well as novel 5b-TP showed moderate inhibition of pol-α (58-76%
inhibition at 100 M) while other NTPs did not or only weakly inhibited pol-α. The moderate
inhibition by 3a-TP, 5c-TP, and 5b-TP may have resulted from the replacement of 2’-OH by 2’-
F as all the three nucleotides have 2’-fluoro on the α-face, which might have moderately reduced
the discrimination by pol-α. All the NTPs tested had little inhibition on pol-β and pol-. All the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NTPs tested showed IC values of >100 M in the polymerase inhibititon assay against human
5
0
RNA pol II. In an assay to investigate the nucleotide incorporation by HMRP (Figure 4), only the
known 2’-C-methyl-UTP (1a-TP) and 2’-C-chloro-UTP (4a-TP) showed weak, but appreciable
incorporation while all the other NTPs tested including 2a-TP and 4b-TP showed basically no
incorporation. Overall, the results indicate that the 4’-F NTPs as well as the 4’-H NTPs are not
effective inhibitors of human polymerases. In certain cases, 4’-F NTPs seem more selective. Thus,
2
a-TP was basicallly not incorporated into RNA by HMRP while 1a-TP was appreciably
incorporated. Based on the promising viral and human polymerase inhibition results, the
nucleotide prodrugs of the 4’-F nucleosides together with some 4’-H analogues were evaluated in
cell based assays against the HCV subgenomic replicon.
Table 2. Inhibition of human polymerases by nucleoside triphosphates
hPol-α
hPol-β
hPol-
% inhibition
@ 100 M
14.6
hRNA pol II
NTP
% inhibition
@ 100 M
% inhibition
@ 100 M
IC50, M
1
a-TP
2a-TP
3a-TP
22.4
16.1
58.2
-0.0080
12.6
>100
>100
>100
9.6
14.0
9.2
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5
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8
9
3
b-TP
a-TP
b-TP
c-TP
5b-TP
40.9
34.5
19.7
70.8
75.8
15.2
22.6
24.3
24.7
18.1
-10.0
9.5
>100
>100
>100
>100
ND
4
4
18.4
15.7
7.9
5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2 3 4 5 6 7 8 9 10 11 12
Figure 4. Nucleotide incorporation by human mitochondrial RNA polymerase
HCV replicon activity
Selective nucleosides were converted to their phosphoramidate prodrugs 44-52 and tested in an
2
8
HCV subgenomic replicon system. As shown in Table 3, the inhibition of HCV replicon by these
phosphoramidate prodrugs correlates well with the inhibitory effects of the corresponding NTPs
in the HCV polymerase assay. Prodrugs of 3b and 4b that have F at the 4’ position have
comparable, potent replicon activity (49 and 51: 0.31 and 0.10 M, respectively) as prodrugs of
3
a, and 4a that have H at the 4’ position (48 and 50: 0.22 and 0.12 M, respectively). As expected
from HCV polymerase inhibition data, the prodrugs 46 and 47 are only weakly active. The other
three 4’-F urdine prodrugs 44, 45 and 52 are highly potent with EC values ranging from 0.045 to
5
0
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
.11 M. Again, the 4’-fluorouridine analogues proved to be highly potent in the cell-based HCV
assay.
Table 3. Inhibition of the subgenomic HCV replicon (genotype 1b) by the prodrugs of 2′-C-
substituted uridines and 4’-fluoro-2’-C-substituted uridines
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
NH
O
O O P O
NH R4
HO
O
N
R
R2
R2'
O
O
Prodrug (of
nucleoside)
EC50
M
CC50
M
R2
R2’
R4
R
methyl
ethynyl
vinyl
OH
OH
OH
OH
F
F
F
F
F
H
F
H
F
F
4
4 (of 2a)
45 (of 2c)
6 (of 2d)
47 (of 2e)
8 (of 3a)
49 (of 3b)
0 (of 4a)
51 (of 4b)
2 (of 5b)
i-Pr
c-hexyl
i-Pr
0.11
0.07
8.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
ethyl
i-Pr
43
methyl
methyl
methyl
methyl
Cl
4
i-Pr
0.22
0.31
0.12
0.10
0.045
F
i-Pr
Cl
Cl
F
5
i-Pr
i-Pr
5
i-Pr
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 at a which 50% of the cells
died.
The nucleoside 2a was selected for prodrug development as shown in Table 4. The two P-isomers
of the prodrug 44 were separated by HPLC to give 53 (Sp) and 54 (Rp). The Sp-isomer 53 (EC50
of 0.07 M) is 13 fold more active than Rp-isomer 54 in the HCV replicon assay. The prodrugs
5
5 and 56 where isopropyl was replaced by cyclohexyl and neopentyl, respectively, are more
potent than 44 in the assay, with IC of 0.04 M for both prodrugs. Likewise, when Ph was
5
0
replaced by naphthyl and p-chlorophenyl, the resulting prodrugs 57 and 58 are also very potent,
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with EC of 0.02 and 0.08 M, respectively. However, the bis-phosphoramidate prodrugs 59 and
5
0
6
0 are only moderately active. None of the prodrugs showed appreciable cytotoxicity in Huh-7
cells. To assess potential cytotoxicity the prodrug 53 was tested in multiple cell lines for 8 days.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
As shown in Table 5, the compound did not show any significant cytotoxicity and CC values are
5
0
>
96 M for all 6 cell lines, including the highly sensitive U937 and MT-4 cell lines. In contrast,
INX-08189, a phosphoramidate prodrug of 2’-C-methyl-6-O-methylguanosine, showed potent
inhibition on several cell lines.
Table 4. HCV replicon activity of 2’-methyluridine 5’-phosphoramidate prodrugs and 4’-fluoro-2’-
methyluridine 5’-phosphoramidate prodrugs
H
H
O
O
O
N
O
O
N
O
Ar
O
O
O
N
R
HN
P
O
N
O O P O
O
O
R
NH
F
NH
F
O
R
HO
OH
O
HO
OH
A
B
Prodrugs of 2a
4 (53+54)
P-chirality
achiral
Chiral, Sp
Chiral, Rp
achiral
achiral
achiral
achiral
NA
Scaffold
Ar
>100
Ph
R
EC50, M
0.11
0.07
0.94
0.04
0.04
0.02
0.08
7.5
CC50, M
>100
4
A
A
A
A
A
A
A
B
B
i-Pr
i-Pr
i-Pr
5
5
5
5
5
5
5
3
4
5
6
7
8
9
>99
Ph
>100.00
84
Ph
c-hexyl
Ph
neopentyl
i-Pr
>100.00
>85
Naph
p-ClPh
i-Pr
>100.00
>100.00
>100.00
--
--
Et
60
NA
i-Pr
6.6
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2
2
2
2
2
2
2
2
3
3
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3
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3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
Table 5. Eight-day cytotoxicity (CC50, M) of compound 53 in six cell lines
Cell Line
Huh-7
96.1
HepG2
>100
1.5
A549
>100
7.8
HeLa
>100
13.4
U937
>100
0.17
MT-4
>100
0.96
5
3
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INX-08189
0.35
Based on its potent activity in the HCV GT-1b replicon and favorable cytotoxicity profile, the
phosphoramidate prodrug 53 was further tested for inhibition of other HCV genotypes. Table 6
shows that the compound is highly potent in all genotypes tested (GT1b, GT1a, GT2b, GT3a and
GT4a) with EC values ranging from 0.04 to 0.06 M.
5
0
Table 6. HCV pan-genotypic replicon activity of compound 53
GT1b WT
0.04 ± 0.02
GT1a
GT2b
GT3a
GT4a
EC50 (µM)
0.06 ± 0.01
0.04 ± 0.01
0.06 ± 0.01
0.06 ± 0.01
Compound 53 is more active than the known compound sofosbuvir (0.07 M vs 0.22 M) in the
HCV replicon assay and its NTP (2a-TP) is slighly more potent (0.14 M vs 0.27 M) than the
TP of sofosbuvir (3a-TP). Compound 53 also demonstrated a very good in vitro safety profile as
shown by the human polymerase inhibition data and 8-day cytotoxicity data. With all these
favorable properties, the compound 53 was selected for further evaluation.
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9
In vitro and in vivo NTP formation
NTP levels in the target tissue are important to 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 level. For compound 53, we measured the in vitro NTP
levels in two cell types, primary human hepatocytes and Huh-7 cells. As shown in Table 7, high
levels of NTP were formed in human hepatocytes and Huh-7 cells following an incubation of 53
at 50 µM extracellular concentration for 24 hours. In human hepatocytes, the equilibrium between
NTP formation and degradation was typically reached between 6 and 24 hours (Figure 5). 2a-TP
intracellular concentrations increased approximately dose proportionally in the dose range of 0.3
to 30 µM and greater than dose proportionally from 30 to 50 µM of 53 incubation concentrations
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 6). Following a 24-hr incubation of 44 (~1:1 mixture of 53 and 54) in human hepatocytes,
high concentrations of NTP were formed. Once formed and after the removal of 44 from the
incubation media, the NTP levels were maintained for 6 hours before degradation took place
(Figure 7). The mean intracellular half-life of the NTP was thus estimated as 30.1 ± 6.3 hours (n
=
3) in human hepatocytes.
In vivo NTP formation was evaluated in beagle dogs following a single oral administration of 53
at 9.87 mg/kg (5 mg/kg parent nucleoside equivalent dose). As shown in Table 7, liver NTP
concentrations were 23190, 35222, and 29991 nM at 4, 12, and 24 hours post dose, respectively.
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3
2
00
00
00
00
0
1
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7
8
9
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
100
0
0
20
40
Time (hr)
60
80
Figure 5. Time course of intracellular NTP in primary
human hepatocytes following incubation with 53 at 10 µM for
2 to 72 hours
3
2
2
1
1
000
500
000
500
000
5
00
0
0
10
20
30
40
50
60
AL-335 Extracellular Concentration (µM)
Figure 6. Intracellular NTP concentration-extracellular 53
concentration response in primary human hepatocytesa
a
Data were collected from hepatocytes from three
different human donors and express as mean ± SD. Each
experiment was conducted with 24 hours of incubation of
5
3 with hepatocytes.
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1
000
1
00
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10
0
4
8
12
16
20
24
28
32
36
40
44
48
Time (hr)
Figure 7. Intracellular NTP concentration-time profile in
primary human hepatocytes following an initial 24-hour
incubation with 44 at 50 µM.
Table 7. NTP formation in vitro and in vivo
Compound
NTP (pmol/Million Cells)a
Dog Liver NTP (nM)b
12 hr
Human
Huh-7
Hepatocytes
4 hr
24 hr
5
3
3540 ± 3133
5695 ± 1793
23190
35222 ± 10478
29991 ± 11873
a
b
Results were obtained after incubation at 50 µM of the compound for 24 hours.
Results were obtained following oral administration of compound 53 at 9.87 mg/kg to dogs, N=2 at 4 hr and N=3 at
1
2 and 24 hr.
CONCLUSSIONS
we have synthesized a series of novel uridine analogues with 4’-fluor-2’-substituted sugar
moieties. Several of the 4’-fluorouridine 5’-triphosphates demonstrated potent inhibition in the
HCV polymerase NS5B assay with IC values as low as 27 nM and showed little inhibition of
5
0
human DNA and RNA polymerases. The 5’-triphosphate of 2a was confirmed to be a chain
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
terminator in a chain elongation assay using the HCV NS5B polymerase. Phosphoramidate
prodrugs of these nucleosides exhibited very potent HCV subgenomic replicon activity with EC50
values as low as 20 nM. Compared with the well-known nucleoside 3a (parent nucleoside of
sofosbuvir), some of the 4’-fluoro-2’-substituted uridines demonstrated equipotent or more potent
in vitro activities as shown by their 5’-triphosphate and prodrugs. All the prodrugs tested exhibited
little cytotoxicity in a 3-day cytoxcity assay. A lead compound 53 demonstrated an excellent safety
profile in an 8-day cytotoxicity assay using six cell lines with all CC values >96 M. Compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
5
3 was also highly potent in all the HCV replicon of 4 genotypes (GT-1b, 1a, 2b, 3a, 4a).
Compound 53 showed high levels of TP of its parent nucleoside (2a-TP) in both in vitro and in
vivo studies, and the NTP has a half-life of approximately 30 hours. The high levels of the NTP in
vivo and long half-life indicate that a once daily therapy may be appropriate. Due to its excellent
in vitro and vivo properties, compound 53 (AL-335) was advanced into clinical development
where promising results were observed in human Phase 1 and 2 clinical trials.13
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 all
non-salt compounds were >95% as determined on an Agilent 1200 HPLC, XTerra 3.5µm
4
.6x150mm MS C18 column, using 0.04 (v/v) TFA in water and 0.02 (v/v) TFA in acetonitrile as
mobile phase. The purities of all nucleotides were >95%, determined on an Agilent 1100 HPLC,
1
19
13
5
0 mM TEAA in water and 50 mM TEAA in acetonitrile as mobile phase. H-, F and C-NMR
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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 s (singlet), d (doublet), t (triplet), q
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(quartet), m (multiplet), or br s (broad singlet). Coupling constants (J) are reported in herz (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, Phenomenex Luna
C18 5µm 5.0x20mm column; mobile phase: 0.04%(v/v) TFA in water and 0.02%(v/v) TFA in
o
acetonitrile, 40 C, flow rate 0.4 mL/min. Mass spectrometric analyses for nucleotides were
performed on an Agilent 1100 HPLC with API 2000 LC-MS/MS System using ESI as ionization,
Synergi 75x2.0 mm, 4µm Hydro-RP80Å column, 50 mM TEAA in water and 50 mM in
acetonitrile, flow rate 0.4 mL/min. Work-up procedures for most of chemical reactions are the
same or similar, therefore, unless specifically indicated, the work-up refers to the following
o
procedure: the reaction mixture at 0 C 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. Unless necessary, the word “work-up” will
not be mentioned for a reaction if it follows the procedure described above. Purification on silica
gel refers to a flash chromatography on a silica gel column.
3
2
’-C-Hydroxymethyl-2’,3’,5’-tri-O-benzyl-N -(4-methoxybenzyl)uridine (9). To a solution of
1
4
o
benzoylated nucleoside 6 (50 g, 86 mmol) in DMF at 0 C were added PMBCl (16 g, 0.1 mol)
and K CO (17.8 g, 0.13 mol). After stiring at rt for 12 h, the reaction mixture was quenched with
2
3
water (100 mL), and extracted with EtOAc (3x200 mL). The organic phase was evaporated to solid
residue which was dissolved in MeOH/DCM (4/1, v/v, 200 mL). The resulting solution was treated
with NaOMe (16.8 g, 0.31 mol), stirred at rt for 2.5 h, and then quenched with dry ice. Usual work-
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
up followed by purification on silica gel column with 1% MeOH in DCM yielded nucleoside 7 as
+
yellow foam (25 g, 75%). MS, m/z 391.1 [M+1] . To a solution of 7 (25.5 g, 65 mmol) in DMF at
o
0 C was added slowly NaH (60%, 10.5 g, 260 mmol) and the resulting mixture was stirred for 30
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
min. Then BnBr (36.3 g, 0.21 mol) was added and the reaction mixture was stirred at rt for 12 h.
Reaction was quenched with saturated NH Cl (aq.). Usual work-up and purification on silica gel
4
column using 10% EtOAc in hexanes yielded benzylated nucleoside 8 (20 g, 46%) as a white solid.
+
MS, m/z 661.2 [M+1] . To a solution of 8 (20 g, 30 mmol) and NMMO (7 g, 60 mmol) in THF/H O
2
(
5/1) (100 mL) was added OsO (2.6 g, 10 mmol). The resulting mixture was kept for 24 h at rt
4
and quenched with saturated aq. Na S O . After usual work-up, the crude diol was dissolved in
2
2
3
MeOH/H O/THF (170 mL/30 mL/50 mL), followed by addition of NaIO (9.6 g, 45 mmol), and
2
4
the resulting mixture was stirred at rt for 2 h. Reaction mixture was filtered and NaBH (1.8 g, 48
4
o
mmol) was added portionwise to the filtrate at 0 C. The resulting mixture was stirred at rt for 30
min. Ususal work-up and chromatography on silica gel column using 20% EtOAc in hexanes gave
2 g of 9 (61%). MS, m/z 665.2 (M + 1)+.
1
2
’-C-Fluoromethyluridine (11). To a solution of hydroxymethyl nucleoside 9 (14 g, 21 mmol)
o
and DMAP (5.1 g, 42 mmol) in DCM (100 mL) at 0 C was added MsCl (3.1 g, 27 mmol). The
resulting solution was stirred at rt for 40 min and then quenched with saturated aq. NaHCO . Usual
3
work-up and purification on silica gel column with 5% EtOAc in hexanes yielded 14 g (90%) of
the mesylate. The mesylate (41 g, 55 mmol) was dissolved in 1.0 M TBAF in THF (500 mL) and
o
stirred at 70-80 C for 3 days when LCMS showed that half of the starting material was converted
to the desired product. Usual work-up and purification on silica gel column using 10% EtOAc in
+
hexanes afforded 9.9 g (27%) of fluoromethyl nucleoside 10. MS, m/z 667 (M + 1) . Obtained
compound 10 (6.3 g, 9.45 mmol) and CAN (15.5 g, 28.3 mmol) was stirred overnight at rt. Usual
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9
work-up and chromatography on silica gel using 20% EtOAc in hexanes gave N-deprotected
compound (3.6 g, 71%) as a white solid. To a solution of the product from previous step (2.4 g,
4.4 mmol) in anhydrous DCM (10 mL) was added dropwise BCl (1 M in DCM, 30 mL) at -70
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
oC. The reaction mixture was stirred at the same temperature for 2 h and then quenched with
MeOH. Usual work-up and purification on silica gel using 50% EtOAc in hexanes afforded 1112
(
1.05 g, 86%) as a white solid. MS, m/z 276.9 (M + 1)+.
5’-Deoxy-5’-iodo-2’-C-(fluoromethyl)uridine (12). To a solution of PPh (3.37 g, 12.8 mmol) in
3
o
pyridine (15 mL) at 0 C was added iodine (3.06 g, 12 mmol). The mixture was stirred at rt for 30
o
min and then cooled to 0 C when solution of nucleoside 11 (2.2 g, 8 mmol) in pyridine (5 mL)
was added. The reaction mixture was stirred overnight at rt and then quenched with saturated aq.
Na S O . Usual work-up and chromatography on silica gel using 1-2% MeOH in DCM gave 12
2
2
3
(
1.8 g, 58%) as a white solid. MS, m/z 387.0 (M + 1)+.
1
-((2R,3R,4S)-3-(Fluoromethyl)-3,4-dihydroxy-5-methylenetetrahydrofuran-2-
yl)pyrimidine-2,4(1H,3H)-dione (13). A solution of iodide 12 (1.35 g, 3.5 mmol) and DBU (1.06
o
g, 7 mmol) in THF/CH CN (2/1, v/v, 35 mL) was stirred at 60-70 C for 2 h. The reaction mixture
3
was concentrated and then dissolved in EtOAc (20 mL), washed with 10% aqueous HCl, brine and
dried (MgSO ). The evaporated residue was purified on silica gel using 30% EtOAc in hexanes to
4
give olefin 13 (0.5 g, 55%) as a foam. MS, m/z 259.1 (M + 1)+.
’-Deoxy-5’-iodo-4’-fluoro-2’,3’-di-O-benzoyluridine (16b). To a stirred solution of olefin 1418
5
o
(
12.0 g, 53 mmol) in anhydrous CH CN at 0 C was added TEA-3HF (8.5 g, 53 mmol) and NIS
3
o
(
10.2 g, 63.6 mmol). The reaction mixture was stirred for 30 min at 0 C and then another 30 min
at rt. Precipitate was removed by filtration and washed with DCM. The filtrate was concentrated
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to give 5’-iodo-4’-fluoro uridine analogue (14 g, 73%) as a yellow solid. MS, m/z 373.0 (M + 1)+.
A solution of the compound prepared in previous step (12.0 g, 32 mmol), Bz O (21.7 g, 96 mmol),
2
o
and DMAP (1.2 g, 9.6 mmol) in pyridine (100 ml) was stirred at 50 C overnight. The reaction
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mixture was quenched with water. Usual work-up and purification on silica gel column with 50%
EtOAc in hexanes to yielded 16b (15 g, 81%) as a white solid. MS, m/z 581.0 (M + 1)+.
5
’-Deoxy-5’-iodo-4’-fluoro-2’,3’-di-O-acetyl-2’-C-methyluridine (16c). A solution of 1519
(12.0 g, 50 mmol) in pyridine (200 mL) and acetic anhydride (14.2 mL, 150 mmol) was stirred 1d
at rt. Reaction was quenched with MeOH, concentrated and the residue coevaporated with toluene.
Usual work-up and column chromatography with 50% EtOAc in hexanes yielded diacetate (15.0
g, 91%) as a colorless foam. To an ice-cold solution of the diacetate (15.0 g, 46.3 mmol) in
anhydrous DCM (300 mL) were added AgF (29.4 g, 231 mmol) and a solution of iodine (23.5 g,
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2.6 mmol) in anhydrous DCM (1.0 L). The reaction mixture was stirred at rt for 5 h, then
quenched with sat. aq. Na S O and NaHCO . Usual work-up and purification on a silica gel with
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2
3
3
1
10-30% EtOAc in hexanes gave 16c (9.5 g, 44%) as a white solid; H NMR (CD OD) δ 7.52 (d, J
3
=
8.0 Hz, 1H), 6.21 (s, 1H), 5.80 (d, J = 17.2 Hz, 1H), 5.73 (d, J = 8.0 Hz, 1H), 3.58 (s, 1H), 3.54
(d, J = 6.8 Hz, 1H), 2.17 (s, 3H), 2.09 (s, 3H), 1.58 (s, 3H).
4
’-Fluoro-2’,3’-di-O-benzoyluridine (17b). Tetrabutylammonium hydroxide (288 mL, 54-56%
aqueous solution, 0.58 mol) was adjusted to pH 4 by adding TFA (48 ml). The resulting solution
was added to a solution of 16b (14 g, 24 mmol) in DCM (200 ml). m-Chloroperbenzoic acid (30
g, 60-70%, ~120 mmol) was added in portions under vigorous stirring and the resulting reaction
mixture was stirred overnight. Usual work-up and chromatography on silica gel with 4% MeOH
in DCM gave 17b (7.5 g, 54%) as off-white solid.
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’-Fluoro-2’-O-acetyl-3’-5-di-O-benzoyl-2’-C-methyluridine (17c). A mixture of 16c (7.0 g,
1
4.9 mmol), NaOBz (21.44 g, 149 mmol) and 15-crown-5 (32.75 g, 149 mmol) in anhydrous DMF
o
(400 mL) was stirred at 130 C for 6 h and then concentrated. Usual work-up and purification on
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a silica gel with 10-30% EtOAc in hexanes gave 17c (2.8 g, 41%). H NMR (CDCl ) δ 8.84 (s,
3
1
1
H), 8.04-8.06 (m, 2H), 7.59 (t, J = 7.2 Hz, 1H), 7.44-7.47 (m, 2H), 7.21-7.26 (m, 1H), 6.21 (s,
H), 5.85 (d, J = 18 Hz, 1H), 5.67 (d, J = 8.0 Hz, 1H), 4.59-4.72 (m, 2H), 2.14 (s, 6H), 1.64 (d, J
=
6.0 Hz, 3H). MS, m/z 527.2 (M + 1) +.
4
’-Fluorouridine (18). A solution of 17b (5.0 g, 8.7 mmol) in methanolic ammonia (7 M, 100
mL) was stirred at rt for 5 h and then concentrated. The solid crude product was filtered and washed
1
with DCM to give 18 (2.1 g, 92%) as white foam. H-NMR (CD OD)  = 7.71 (d, J = 7.6 Hz, 1H),
3
6
.05 (s, 1H), 5.68 (d, J = 7.6 Hz, 1H), 4.39-4.45(m, 1H), 4.25 (d, J = 7.6 Hz, 1H), 3.72-3.73(m,
2
H). MS, m/z 263.0 (M + 1)+.
3
2
’,3’-di-O-Benzoyl-2’-C-ethynyl-4’-fluoro-N -benzoyluridine (25). A solution of TsOH·H O
2
(0.7 g, 3.7 mmol), 1,1-dimethoxycyclopentane (19.3 g, 148.5 mmol) and 2’-ethynyluridine 1c14
o
(10.0 g, 37.1 mmol) in DCE (100 mL) was stirred at 50 C overnight. The reaction mixture was
neutralized with Et N and concentrated. The residue was purified on silica gel (1-10% MeOH in
3
+
DCM) to give 21 (8.7 g, 69% ) as a white solid. MS, m/e 335.0 (M+1) . To an ice-cold solution
of 21 (20.0 g, 0.06 mol) in anhydrous pyridine (100 mL) was added TsCl (22.8 g, 0.12 mol). The
reaction mixture was stirred overnight, then quenched with water. A usual work-up and
purification on silica gel (1-6% MeOH in DCM) produced 5’-O-tosyl nucleoside (20.0 g, 69%) as
a white solid. A solution of tosylate (20.0 g, 0.04 mol) and NaI (31.0 g, 0.2 mol) in acetone (200
mL) was refluxed overnight and then quenched with saturated Na S O solution. Usual work-up
2
2
3
and chromatography on silica gel (1-6% MeOH in DCM) gave iodide 22 (15.0 g, 56%) as a white
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solid. MS, m/e 443.1 (M-1) . Compound 22 (13.4 g, 30.2 mmol) in 50 ml of 80% aq. AcOH was
o
stirred at 60 C for 2 h. The reaction mixture was concentrated and the residue purified on silica
gel (1-10% MeOH in DCM). The product (5.0 g, 13.22 mmol) was treated as described for 13 to
give the olefin 23 as a white solid. After iodofluorination of 23, as described for 16b, with
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subsequent benzoylation the product 24 (1.8 g, 19 %) was obtained as a white solid. MS, m/z
+
7
09.1 (M+1) . Compound 24 (600 mg, 0.85 mmol) in DCM (10 mL) yielded 25 (123 mg, 24%)
as a white solid as described for 17b. MS, m/z 599.1 (M + 1)+.
(
4S,5R)-ethyl-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-4-methyl-1,3,2-dioxathiolane-4-
2
1
carboxylate 2,2-dioxide (29). TBACl (102.5g, 370 mmol) was added to a solution of 28 (30.0
o
g, 95 mmol) in anhydrous dioxane (1 L), and the mixture was heated at 120 C overnight. After
cooling to rt. 2,2-dimethoxypropane (300 mL) and conc. hydrochloric acid (20 mL) were added.
The mixture was stirred for 3 h at rt, then evaporated to 1/3 of the volume, diluted with EtOAc and
then washed with cold saturated sodium bicarbonate and brine. The combined aqueous layer was
extracted with EtOAc (2 x 100 mL). The combined organic extract was dried (Na SO ), filtered,
2
4
1
and concentrated to give 29 (22 g, 71%). H-NMR (DMSO-d ), δ 4.18-4.03 (m, 2H), 3.90-3.82(m,
6
1
H), 3.15-3.32 (m, 2H), 1.50-1.57 (m, 2H), 1.18-1.40 (m, 9H), 0.92-0.96 (m, 3H).
1
-O-Acetyl-2-deoxy-2-chloro-3,5-di-O-benzoyl-2-C-methyl-α/β-D-ribofuranose (31). The
ester 29 (22 g, 82.7 mmol) was dissolved in ethanol (200 mL) and conc. hydrochloric acid (6 mL).
The resulting solution was stirred at rt for 48 h, then concentrated under reduced pressure, the
residue co-evaporated with toluene to give the unprotected lactone as a pale yellow solid (10.7 g).
The unprotected lactone was dissolved in anhydrous pyridine (100 mL) and benzoyl chloride (20
o
mL, 167 mmol) was added dropwise at 0-5 C. The resulting mixture was stirred at rt for 45 min,
then quenched with ice and MeOH to form a suspension. The solid was filtered and washed with
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MeOH to give ribonolacton 30 (18.5 g, 56.6% ) as white solid. The lactone 30 (2.3 g, 5.9 mmol)
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was dissolved in anhydrous THF (50 mL) and the solution was cooled to -60 C under nitrogen.
Lithium tri(tert-butyl)aluminum hydride (1.0 M in THF, 20 mL) was added over 3 min with
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stirring at -60 C. After 3 h at the same temperature, the mixture was quenched and diluted with
saturated aqueous NH Cl (30 mL). Resulting precipitate was filtered and washed with EtOAc (3
4
x 50 mL). The combined organic extract was concentrated and re-dissolved in THF (50 mL).
o
DMAP (80 mg, 0.66 mmol) and acetic anhydride (5.46 g, 53.5 mmol) were added at 0 C. The
o
mixture was stirred at 0-5 C for 1 h, concentrated, and the residue was purified on silica gel with
1
3
5% EtOAc in hexanes to give acetate 31 (2.0 g, 79%) as a white solid. H-NMR (CDCl ), δ 7.54-
3
8.11 (m, 10H), 6.35 (s, 1H), 5.78 (d, J = 8.0 Hz, 1H), 4.65-4.74 (m, 2H), 4.43 (dd, J = 11.6 Hz,
4
.4Hz, 1H), 1.97 (s, 3H), 1.69 (s, 3H).
4
2
’-Deoxy-2’-chloro-2’-C-methyl-3’,5’-di-O-benzoyl-N -benzoylcytidine (32). A suspension of
4
N -benzoylcytosine (2.3 g, 11 mmol) and ammonium sulfate (45 mg) in hexamethyldisilazane (30
mL) was refluxed for 4 h and the resulting solution was concentrated, followed by drying under
vacuum for 3 h. The resulting silylated cytosine was dissolved in chlorobenzene (30 mL). To this
stirred solution was added riboside 31 (2.0 g, 4.6 mmol) and tin(IV) chloride (2.4 mL, 10.5 mmol).
o
After stirring at rt for 2 h, the reaction mixture was heated at 60-70 C overnight, then cooled to 0
oC, and quenched with solid sodium bicarbonate (4 g) , followed by slow addition of water (20
mL). In 20 min precipitate was filtered and washed with EtOAc. Usual work-up and purification
by column chromatography (5-35% EtOAc in hexanes) afforded nucleoside 32 (1.2 g, 44.6%) as
1
a white solid. H-NMR (CDCl ), δ 8.82 (br, s, 1H), 7.56-8.22 (m, 17H), 6.72 (s, 1H), 5.59 (d, J =
3
8
.8 Hz, 1H), 4.86-4.90 (m, 1H), 4.78-4.82 (m, 1H), 4.64 (dd, J = 13.2 Hz, J =3.2 Hz, 1H), 1.61
1 2
(
s, 3H). MS, m/e 588 (M+1)+.
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