Synthesis of 9-(2-β-C-methyl-β-d-ribofuranosyl)-6-substituted purine derivatives as inhibitors of HCV RNA replication

Article abstract of DOI:10.1016/j.bmcl.2004.11.020

A series of 9-(2′-β-C-methyl-β-d-ribofuranosyl)-6- substituted purine derivatives were synthesized as potential inhibitors of HCV RNA replication. Their inhibitory activities in a cell based HCV replicon assay were reported. A prodrug approach was used to

Full text of DOI:10.1016/j.bmcl.2004.11.020

Bioorganic & Medicinal Chemistry Letters 15 (2005) 709–713
Synthesis of 9-(2-b-C-methyl-b-D-ribofuranosyl)-6-substituted
purine derivatives as inhibitors of HCV RNA replication
Yili Ding,* Jean-Luc Girardet, Zhi Hong, Vicky C. H. Lai, Haoyun An, Yung-hyo Koh,
Stephanie Z. Shaw and Weidong Zhong
Valeant Pharmaceuticals International, 3300 Hyland Avenue Costa Mesa, CA 92626, USA
Received 20 August 2004; revised 1 November 2004; accepted 6 November 2004
Available online 30 November 2004
Abstract—A series of 9-(20-b-C-methyl-b-D-ribofuranosyl)-6-substituted purine derivatives were synthesized as potential inhibitors
of HCV RNA replication. Their inhibitory activities in a cell based HCV replicon assay were reported. A prodrug approach was
used to further improve the potency of these compounds by increasing the intracellular levels of 50-monophosphate metabolites.
These nucleotide prodrugs showed much improved inhibitory activities of HCV RNA replication.
Ó 2004 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infection is the leading cause of
post-transfusion, non-A and non-B viral hepatitis.1
About 170 million people worldwide are thought to be
infected with this virus of which an estimated 4 million
reside in the United States.2 HCV infection causes sig-
nificant liver diseases such as cirrhosis and may eventu-
ally lead to the development of hepatocellular
carcinoma. Currently approved drugs for the treatment
of hepatitis C are combinations of interferon-a and riba-
virin, which result in a sustained virological response
(SVR) in only ꢀ50% of the treated patients and cause
significant side effects in some cases.3 Therefore, the
development of more effective and HCV-specific antivi-
ral agents is an urgent public health need.
showed a greater potency than 20-O-methyl cytidine in
inhibiting HCV RNA replication. However, 20-b-C-
methyl adenosine is a good substrate of adenosine
deaminase, which has prevented this compound from
further development. These interesting results prompted
us to synthesize different types of 9-(2-b-C-methyl-b-D-
ribofuranosyl)-6-substituted purine derivatives and eval-
uated their activities in the HCV subgenomic replicon
assay.
In this letter, we report the synthesis of 9-(2-b-C-
methyl-b-D-ribofuranosyl)-6-substituted purine de-
rivatives including 6-aromatic substituted, sulfur
substituted, N-substituted and O-substituted nucleo-
sides. These compounds were tested as inhibitors of
HCV RNA replication in a cell-based replicon assay.
Previous screening of available nucleoside collections in
the HCV replicon assay has identified two nucleoside
analogs, 20-b-C-methyl adenosine and 20-O-methyl cyti-
dine, which inhibit HCV RNA replication without sig-
nificant cytotoxicity.4 Lower level of the intracellular
20-O-methyl cytidine triphosphate was detected than
that of the 20-b-C-methyl adenosine triphosphate, sug-
gesting that 20-b-C-methyl adenosine was phosphory-
lated more efficient than 20-O-methyl cytidine in the
cells. Consistent with this, 20-b-C-methyl adenosine
Our synthetic strategy is summarized in Scheme 1. By
using an established method,5 we were able to obtain
the key intermediate 2 from the coupling of 1,2,3,5-tet-
ra-O-benzoyl 2-b-C-methyl-D-ribofuranose (1) and 6-
chloropurine in the presence of TMSOTf. Starting from
compound 2, we were able to synthesize 9-(2-
b-C-methyl-b-D-ribofuranosyl)-6-substituted
derivatives.
purine
Maintaining the amino group at position 6 of the purine
base in the nucleoside is likely required for interaction
with the viral polymerase via hydrogen bonding. Thus,
compound 2 was reacted with 25 different nucleophilic
amino derivatives in ethanol followed by deprotection
Keywords: 2-C-Methyl nucleoside; HCV; Inhibitors.
*
Corresponding author. Tel.: +1 714 5450100; fax: +1 714 6417222;
e-mail: yding@icnpharm.com
Present address: Acon laboratories, Inc., 4108 Sorrento Valley
Boulevard, San Diego, CA 92121, USA.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.020

710
Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 709–713
Cl
N
N
N
Cl
O
1
OBz
BzO
N
O
BzO
N
a
N
+
N
H
N
OBz
BzO
OBz
BzO
2
R
N
b
N
N
N
O
HO
3a-3z
OH
HO
N(CH3)2
a
NHC2H4NH2
b
NHC2CH4CONH2
c
NHOC2H5
e
HN
SMe
d
O
N(OMe)CONHEt
f
O
NHCONHEt
j
HN
NHCH3
HN
O
h
g
i
NHEt
m
N(CH3)OH
n
CH3NC2H4NHCH3
o
N(OCH3)CH3
HN
EtO
k
l
O
S
NH(CH3)OC2H4OH NHOCH3
N(OH)CO2Bn
s
N
N
p
t
q
r
NHOC2H4OH
w
NHC2H4OH
v
N(OH)CONHEt
x
N(CH2CH2OH)2
y
NH
u
CN
R
N
N
N
N
N
N
e
c
N
N
O
O
2
BzO
HO
4a R = OMe
4b R = OEt
4c R = OH
4d R = SMe
6
OBz
f
OH
BzO
HO
d
R
N
R
N
N
N
N
N
N
6a R = CNHNHOH
6b R = CNHOMe
6c R = CSNH2
5a = 2-Furan
5b = 2-Thiophene
5c = 3-Thiophene
N
O
HO
O
HO
g
6d R = CNHNH2
OH
HO
OH
HO
Scheme 1. (a) TMSOTf, BSA, CH3CN, 60°C, 3h; (b) (i) amine, ethanol, 50°C, 10h; (ii) NH3/MeOH or NaCN/MeOH, room temperature, 12h; (c)
NaOMe or NaOEt or NaOH or NaSMe, ethanol, 50°C, 4h; (d) (i) Pd(PPh3)4, K2CO3, toluene, RB(OH)2, 100°C, 12h; (ii) NH3/MeOH, room
temperature, 10h; (e) Pd(PPh3)4, Zn(CN)2, toluene, 80°C, 10h; (f) (i) NH2OH or NaCN/MeOH or H2S; (ii) NH3/MeOH, room temperature, 10h; (g)
NH4Cl, MeOH, 70°C, 15h.
with NH3/MeOH or NaCN/MeOH, to yield products
3a–y. When aryl amines were used, the reactions were
performed at 75°C in ethanol for 12h. In the reaction
with 1,2-ethylene diamine, the disubstituted product 3z
was isolated as a minor product. Treatment of com-
pound 2 with NaOMe, NaOEt, and NaOH in methanol
at 50°C for 4h yielded compounds 4a–b and 20-b-C-
methyl-b-D-ribofuranosyl hypoxanthine (4c), respec-
tively, with concomitant deprotection.
used anticancer drugs thioguanine and 6-mercaptopur-
ine.6 Therefore, we decided to investigate this type of
modification with our 20-b-C-methyl nucleosides. Reac-
tion of compound 2 with NaSMe at 50°C produced
9-(2-b-C-methyl-b-D-ribofuranosyl)-6-thiomethoxyl pur-
ine (4d).
Recently, a class of C-6 aryl purine nucleosides has been
shown to possess cytostatic activity toward T-lympho-
blastoid, He–La, and L 1210 cells, as well as L929 cell
lines.7 In the light of this result, we prepared three 9-
(2-b-C-methyl-b-D-ribofuranosyl) 6-aryl-purine deriva-
Replacement of the oxygen atom at the 6-position of
guanine by a sulfur atom generated clinically widely

Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 709–713
711
tives 5a–c, through the Suzuki–Miyaura cross-coupling
reaction8 of compound 2 with three aromatic boronic
acid derivatives, followed by deprotection with NH3/
MeOH.
Several phosphoramidate and hydroxylamino deriva-
tives of 6-N-substituted 20-b-C-methyl adenosine were
also prepared. The synthetic strategy is summarized in
Scheme 2. Treatment of compound 2 with a 2M solu-
tion of NH3/MeOH at room temperature, gave the
20,30,50-tri-O-benzoyl 20-b-C-methyl adenosine (7),
which was dissolved into dry pyridine and added to
the mixture of 1,2,4-triazole, POCl3, and triethylamine
in acetonitrile. This mixture was stirred at room temper-
ature for 1h. When the reaction mixture was treated
with ethanol, after work up and deprotection with
NaCN/MeOH, diethyloxyl phosphoramidate derivative
8a was obtained in 15% yield. When the reaction
mixture was treated with triethylammonia carbonate,
after work up and deprotection with NH3/MeOH,
The cyano group was then introduced at position 6 of
the purine ring by reacting compound 2 with palladium
tetrakis(triphenylphosphine) and zinc cyanide to yield
compound 6. Compound 6 was subsequently converted
into 6-hydroxyaminoamidine, methoxylmino, thioami-
dine derivatives 6a–c by reactions with NH2OH/H2O,
NaCN/MeOH, and H2S, respectively, followed by
deprotection with NH3/MeOH. The 6-amidine substi-
tuted purine derivative 6d was obtained from the reac-
tion of compound 6b with NH4Cl in methanol.
OEt
O
NH2
P
EtO
NH
N
N
N
N
N
N
N
O
BzO
a
N
O
HO
OBz
BzO
OH
8a
HO
7
b
O
R
P
HN OH
N
N
N
N
O
HO
OH
HO
8b = NH2
8c = OMe
Cl
NHOH
NHOH
N
N
N
N
N
N
N
N
N
N
c
d
N
N
O
2
O
O
BzO
BzO
HO
OBz
OBz
OH
9a
BzO
BzO
HO
9
e
f
ROC
N
RHNOC
N
NOH
N
NOH
N
N
N
O
N
N
O
HO
HO
HO
OH
OH
HO
9b R = Ph
9c R = CH(Me)2
9d R = Et
9f R = OMe
9g R = Et
Scheme 2. (a) (i) POCl3, 1,2,4-triazole, triethylamine, CH3CN, 0°C, 30min; (ii) 7, pyridine, room temperature; 1h; (iii) ethanol, room temperature,
1h; (iv) NaCN, MeOH, 15% overall yield; (b) (i) POCl3, 1,2,4-triazole, triethylamine, CH3CN, 0°C, 30min; (ii) 7, pyridine, room temperature; 1h;
(iii) triethylammonia carbonate, room temperature, 1h; (iv) NH3/MeOH; (c) NH2OH, room temperature, 10h; (d) NaCN/MeOH, room
temperature, 12h; (e) (i) (Me)2CHNCO or EtNCO or PhNCO, CHCl3, 40°C, 10h; (ii) NaCN/MeOH, room temperature, 12h; (f) (i) TMSI, 1,2,4-
triazole, pyridine, room temperature, 4h; (ii) C2H5COCl or CH(OMe)3, pyridine, room temperature, 10h; (iii) NH4OH.

712
Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 709–713
Table 1. Inhibitory potency and cytotoxicity of compounds 3a–z, 4a–
d, 5a–c, 6a–d, 8a–c, 9a–g, and 10a–b in HCV replicon assay
2-hydroxyamino phosphoramidate derivative 8b was
isolated in 18% yield, and 2-hydroxymethoxyl phospho-
ramidate derivative 8c was isolated in 5% yield.
Compounds
EC50, lMa Cytotoxicity CC50, lMb
3a
3b
9
25
150
300
30
80
300
300
1
300
300
300
300
300
300
300
300
300
300
300
250
300
300
300
300
300
300
300
200
300
300
300
300
300
300
300
300
300
300
300
200
300
300
300
300
300
300
300
300
300
300
300
200
250
300
300
300
300
Treatment of compound 2 with hydroxylamine at room
temperature for 10h produced compound 9, and after
deprotection with NaCN/MeOH, 6-N-hydroxylamine
derivative 9a was obtained. When compound 9 was trea-
ted with isopropyl isocyanate, ethyl isocyanate and phen-
yl isocyanate in CHCl3 at 40°C for 10h, followed by
deprotection with NaCN/MeOH and purification on sil-
ica gel column, only monosubstituted hydroxylamine
derivatives 9b,c, and 9d were obtained. Treatment of
compound 9a with propionyl chloride and methyl for-
mate in pyridine in the presence of 1,2,4-triazole and
TMSI yielded compounds 9f and 9g.9
3c
3d
3e
3f
3g
3h
3i
3j
3k
80
33
50
8
3l
3m
3n
3o
4
200
100
80
4
3p
3q
Compounds 3a–z, 4a–d, 5a–c, 6a–d, 8a–c, and 9a–g were
assayed for their ability to inhibit HCV RNA replication
in a subgenomic replicon cell line.10 The potency of
these compounds against HCV replicon is expressed as
EC50. The MTS assay was utilized to determine the
associated cytotoxicity (CC50).11 Table 1 summarize
the HCV replicon activity and cytotoxicity of these com-
pounds. As shown, several of these 20-C-methyl nucleo-
sides showed low micromolar inhibitory activities in the
HCV replicon assay.
3r
3s
20
15
25
60
80
200
20
80
9
3t
3u
3v
3w
3x
3y
3z
4a
4b
4c
13
8
To exert antiviral activity in cell culture, nucleoside
analogs need to be phosphorylated intracellularly into
corresponding triphosphates. These triphosphates are
the active forms of the nucleoside inhibitors. The inhibi-
tory potency of the nucleoside inhibitors should thus
correlate with the levels of the triphosphates formed in
the cells.
4d
5a
11
275
80
300
100
120
120
100
25
6
5b
5c
6a
6b
6c
6d
Therefore, the observed low activity for some of these
compounds in HCV replicon assay may be due to their
poor conversion to the active triphosphate forms by
cellular kinases. Therefore, a prodrug approach that
can deliver nucleoside monophosphates to the cells
may overcome the rate-limiting nucleoside phosphory-
lation step and improve antiviral activity of the com-
pounds.12 As in most cases, monophosphates are
usually converted efficiently into the corresponding
triphosphates.
8a
8b
8c
9a
33
1.8
9b
9c
1.8
6
9d
2.2
3
9f
9g
3.3
0.1
0.08
0.3
10a
10b
To demonstrate this concept with the 20-b-C-methyl
nucleoside derivatives, we chose compounds 4a and 4b
as the starting materials to synthesize their monophos-
phate SATE-prodrugs. The synthetic strategy is shown
in Scheme 3. By using an established method,13 com-
pounds 4a and 4b were coupled with the phosphorami-
dites 11a and 11b in the presence of tetrazole, followed
by oxidation of the resulting phosphites with t-butyl
hydroperoxide in DMF, to produce the 50-mono-
phosphate prodrugs 10a and 10b.14
20-b-C-Methyl adenosine
a Compounds were incubated in cell culture for 48h prior to determi-
nation of the relative amount of HCV replicon RNA using a lucif-
erase reporter assay.
b Compound cytotoxicity was determined by MTS assay on parallel
samples.
bypassing the rate-limiting phosphorylation step, the
activity of this class of nucleoside inhibitors in suppress-
ing HCV RNA replication can be significantly
enhanced.
Compounds 10a and 10b were assayed for their ability
to inhibit HCV RNA replication in the replicon assay.
As shown in Table 1, both of them showed a much im-
proved potency (90- and 180-fold over 4a and 4b,
respectively). This result clearly demonstrated that by
In summary, a series of 9-(2-b-C-methyl-b-D-ribofur-
anosyl)-6-substituted purine derivatives were synthe-

Y. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 709–713
713
OR
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O
N
N
R1
N
S
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O
O
N
+
O
HO
R1
P
S
N
O
OH
HO
11a R1 = t-Bu
11b R1 = Me
4a R = Me
4b R = Et
a
OR
O
R1
N
N
S
O
O
N
N
R1
O
O
P
S
O
O
OH
HO
10a R = Me, R1 = t-Bu
10b R = Et, R1 = Me
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sized and tested for their ability to inhibit HCV RNA
replication in the replicon cells. Several of these adeno-
sine analogs showed low micromolar activity in the rep-
licon assay. In order to increase the intracellular levels of
nucleoside triphosphates, which usually correlate with
the antiviral potency of nucleoside inhibitors, two
SATE-prodrugs of 50-monophosphate nucleoside deriv-
atives were synthesized. These prodrugs showed a much
improved potency in inhibiting HCV RNA replication.
This result indicated that the nucleotide prodrug
approach could be applied to anti-HCV nucleosides,
the same way as it was applied to anti-HIV and anti-
HBV nucleosides.
13. Lefebvre, I.; Perigaud, C.; Pompon, A.; Aubertin, A.-M.;
Girardet, J.-L.; Kirn, A.; Gosselin, G.; Imbach, J.-L. J.
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14. All compounds in this study were characterized by proton
NMR and MS spectroscopic analysis.