Synthesis of 2-C-hydroxymethylribofuranosylpurines as potent anti-hepatitis C virus (HCV) agents

Article abstract of DOI:10.1016/j.bmcl.2006.05.089

On the basis of potent anti-HCV activity of 2′-C-methyladenosine, novel 2′-C-hydroxymethyladenosine analogues 2a-c were synthesized from d-ribose in order to lead to favorable interaction with HCV polymerase. Among compounds tested, adenosine derivative 2

Full text of DOI:10.1016/j.bmcl.2006.05.089

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4190–4194
Synthesis of 2-C-hydroxymethylribofuranosylpurines
as potent anti-hepatitis C virus (HCV) agents
Byul Nae Yoo,a Hea Ok Kim,a Hyung Ryong Moon,b Su Kyung Seol,c
Sung Key Jang,c Kang Man Leea and Lak Shin Jeonga,*
aLaboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
bCollege of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
cDepartment of Life Science, Pohang University of Science and Technology, Kyungbuk 790-784, Republic of Korea
Received 14 April 2006; revised 13 May 2006; accepted 29 May 2006
Available online 14 June 2006
Abstract—On the basis of potent anti-HCV activity of 20-C-methyladenosine, novel 20-C-hydroxymethyladenosine analogues 2a–c
were synthesized from D-ribose in order to lead to favorable interaction with HCV polymerase. Among compounds tested, aden-
osine derivative 2a exhibited potent anti-HCV activity, indicating that the hydroxyl group of 20-C-hydroxymethyl substituent led
to favorable interaction with HCV polymerase.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Hepatitis C is a viral disease which afflicts an estimated
170 million people worldwide.1 Hepatitis C virus (HCV)
was identified in 1989 as the major pathogen responsible
for post-transfusion non-A and non-B hepatitis.2
Chronic infection with HCV can be developed as lethal
liver diseases such as liver cirrhosis and hepatocellular
carcinoma.3
of the most potent inhibitors (EC50 = 0.3 lM) in a cell-
based HCV replicon assay (Fig. 1).9 Its guanosine9
and cytidine10 analogues also exhibited potent anti-
HCV activity in the same assay system. These
nucleosides are incorporated into proviral RNA like a
substrate chain after being converted to their correspond-
ing triphosphates and act as chain-terminators because
20-methyl group prevents subsequent incorporation of
incoming nucleoside triphosphate (NTP).11
However, effective therapeutics are still not available for
the treatment of HCV-infected individuals although
immunotherapy using recombinant interferon-a in com-
bination with ribavirin is being clinically used.4
On the basis of these findings, we designed the 20-C-hy-
droxymethyl analogues 2, which put the additional
hydroxyl group to 20-C-methyladenosine 1, to expect
the favorable electronic effect, in addition to the desired
steric effect by 20-C-hydroxymethyl group on the inter-
action with HCV polymerase (Fig. 1). While synthesiz-
ing the target nucleosides, we encountered that the
electronic effect was more dominant factor than the ste-
ric effect in the glycosylation to obtain b-nucleosides.
Herein, we report the synthesis of 20-C-hydroxymethyl-
adenosine derivatives (2a–c) as anti-HCV agents and
their related interesting sugar chemistry.
HCV belongs to RNA virus with a single strand RNA
genome encoding a 3000 amino acid polyprotein which
is processed into several viral proteins,5,6 including the
NS5b RNA dependent RNA polymerase.7 This enzyme
is essential for viral replication and has been a valid anti-
viral target for the development of new anti-HCV
agents.
A number of nucleoside and non-nucleoside derivatives
have been synthesized and tested for anti-HCV activity.8
Among these, 20-C-methyladenosine (1) emerged as one
Our initial synthetic approach was to synthesize the gly-
cosyl donor 5 and then to condense with nucleobase, as
shown in Scheme 1.
Keywords: Hepatitis C virus; RNA dependent RNA polymerase; 20-C-
Hydroxymethyladenosine; Anti-HCV agent.
D-Ribose was treated with acetone and catalytic amount
of sulfuric acid to give the acetonide 3. Compound 3
was subjected to the aldol condensation to give the
*
Corresponding author. Tel.: +82 2 3277 3466; fax: +82 2 3277
2851; e-mail: lakjeong@ewha.ac.kr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.089

B. N. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4190–4194
4191
silylated thymine in the presence of trimethylsilyl triflu-
oromethane sulfonate (TMSOTf) as a Lewis acid to give
the a-anomer 6 with trace amounts of its b-anomer. In
this glycosylation, the neighboring group effect by the
C2 acetoxymethyl group was found to be more domi-
nant than the steric effect by 2,3-acetonide.
In order to avoid the exclusive formation of a-anomer
through the neighboring group effect by 2-C-acetoxy-
methyl group, 2-C-hydroxymethyl group was converted
to the 2-C-benzyloxymethyl group, as shown in
Scheme 2. The benzyl group was selectively introduced
at the primary hydroxyl group of 4 using organotin
chemistry13 to give the dibenzyl ether 7 as an anomeric
mixture. Compound 7 was acetylated to give another
glycosyl donor 8. However, condensation of 8 with
silylated thymine also afforded the a-anomer 9a as a
major product, indicating that 2-C-benzyloxymethyl
Figure 1. The rationale for the design of the target nucleosides.
2-hydroxymethyl derivative 4 as a single stereoisomer,12
which was peracetylated to give the glycosyl donor 5. As
a model study, the glycosyl donor 5 was condensed with
Scheme 1. Reagents and conditions: (a) acetone, concd H2SO4, rt, 2.5 h; (b) CH2O, K2CO3, MeOH, reflux, 4 d; (c) Ac2O, DMAP, NEt3, rt, 5 h.
Scheme 2. Reagents and conditions: (a) i. n-Bu2SnO, toluene, reflux, 15 h; ii. n-Bu4NI, BnBr, 100 ꢁC, 15 h; (b) Ac2O, pyridine, rt, 8 h; (c) i. thymine,
(NH4)2SO4, HMDS; ii. TMSOTf, ClCH2CH2Cl, overnight; (d) 3 N HCl/THF (1:1), rt, 1 d; (e) Ac2O, Et3N DMAP, CH2Cl2, rt, 3 h.

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B. N. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4190–4194
group is more dominant than 2,3-acetonide from the
viewpoint of stereoelectronic effect. Thus, we decided
to synthesize the glycosyl donor 11 whose C2 hydroxyl
group was protected with acetyl group, for the exclusive
formation of the b-anomer by neighboring group partic-
ipation of the C2 acyl group.
Treatment of 7 with 3 N HCl in THF under reflux for
1 d afforded inseparable anomeric mixture of 10, which
was decomposed when the reaction mixture was evapo-
rated without complete neutralization. Treatment of
compound 10 with acetic anhydride in the presence of
DMAP and triethylamine gave 11a (a-anomer, 55%)
and 11b (b-anomer, 40%), after purification by silica
gel column chromatography. Anomeric configuration
1
of 11a and 11b were readily assigned by H NOE. The
NOE effect (2.94%) was observed between the anomeric
proton of 11a and its methylene protons of 2-CH2OBn,
indicating a-anomer, while no NOE was observed on
the same experiment in the case of 11b, resulting in
b-anomer.
Condensation of the acetate, 11a or 11b with persilylat-
ed 6-chloropurine under Vorbruggen conditions using
TMSOTf yielded the same b-anomer 12 as a single iso-
mer, through the neighboring group effect, as expected
(Scheme 3). Interestingly, compound 11a was rather
unreactive toward condensation with 6-chloropurine,
when compared with compound 11b. In case of 11b,
condensation reaction was completed in 4 h at room
temperature, whereas compound 11a required higher
temperature (50 ꢁC). Treatment of 12 with sodium meth-
oxide gave the desired product 13a and small amounts
of 6-methoxy compound 13b. The stereochemistry of
compounds 12 was established by 1H NOE experiments.
Irradiation on anomeric proton in 12 gave a NOE
(1.26%) on the 40-proton, indicating the b-anomer.
Scheme 3. Reagents and conditions: (a) i. 6-chloropurine, (NH4)2SO4,
HMDS, 160 ꢁC, overnight; ii. TMSOTf, ClCH2CH2Cl; (b) NaOMe,
MeOH, 1 h.
for monitoring anti-HCV activities of compounds. This
system is composed of a human hepatocarcinoma cell
line (Huh-7) supporting multiplication of a HCV repli-
con named NK-R2AN. This replicon consists of the 50
nontranslated region (50NTR) and 30NTR, which are re-
quired for replication of viral RNA, a selection marker
gene neomycin phosphotransferase II and a reporter
Renilla luciferase under the control of the HCV internal
ribosomal entry site (IRES), and the HCV sequence
encoding nonstructural proteins (NS3, 4a, 4b, 5a, and
5b) under the control of the encephalomyocarditis virus
(EMCV) IRES. Antiviral activities of compounds are
reflected by the reduction of Renilla luciferase activity
in the Huh-7 cells. Among compounds tested,
compound 2a exhibited potent anti-HCV activity. Com-
pound 2a inhibited the replication of the replicon
NK-R2AN in Huh-7 cells by 50% at 10 lM concentra-
tion. The replication of the replicon was inhibited
by 2% at 1 lM concentration in the same cell line.
However, other nucleosides did not show any significant
antiviral activity.
The final nucleosides 2a–c were synthesized from the key
intermediate 13a (Scheme 4).
A solution of 13a in methanolic ammonia was heated at
80 ꢁC in a glass bomb for 1 d to give the adenine deriv-
ative 14 in 85% yield. Compound 13a was converted to
N6-methyladenine derivatives 15 (97%) by heating with
40% aqueous methylamine in methanol. Compound
13a was also converted to the hypoxanthine analogue
16 by treatment with 2-mercaptoethanol and sodium
methoxide in methanol under reflux in 78% yield. It is
known that compound 13a was generally converted to
16 via the unstable intermediate 17a, but thioketal 17b
was also formed as a stable minor adduct, as shown in
Scheme 5.
The thioketal 17b was desulfurized to give the same
compound 16 by treating with mercuric chloride and
potassium acetate in acetic acid.14 Treatment of com-
pounds 14–16 with palladium black in 50% formic acid
in methanol afforded the final nucleosides 2a–c,15
respectively.
In summary, on the basis of potent anti-HCV activity of
20-C-methyladenosine (1), we have accomplished the
stereoselective synthesis of b-20-C-hydroxymethyladeno-
All synthesized compounds were tested for anti-HCV
activity using an in vitro assay system that is suitable

B. N. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4190–4194
4193
Scheme 4. Reagents and conditions: (a) NH3/MeOH, 80 ꢁC, 1 d; (b) 40% CH3NH2 in H2O, MeOH, 80 ꢁC, 1 h; (c) 2-mercaptoethanol, NaOMe,
MeOH, 20 h; (d) Pd/black, 50% HCOOH in MeOH, 50 ꢁC, 5 h.
Scheme 5. Reagents and conditions: (a) 2-mercaptoethanol, NaOMe, MeOH; (b) HgCl2, AcOH, KOAc, reflux, overnight.
sine derivatives from D-ribose, utilizing a neighboring
group effect. During the glycosylation, the electronic ef-
fect by acetyl group proved to be more dominant factor
than the steric effect by CH2OBn or isopropylidene
group and CH2OBn side chain had more impact than
isopropylidene group in view of steric effect. Among
compounds synthesized, adenosine derivative 2a exhibit-
ed potent anti-HCV activity, indicating that the hydrox-
yl group of 20-C-hydroxymethyl side chain might have
favorable interaction with HCV polymerase.
Acknowledgment
This research was supported by a grant from Korea
Research Foundation (KRF-2005-005-J01502).

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B. N. Yoo et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4190–4194
CHa), 3.42 (d, 1H, J = 10.4 Hz, 20-hydroxymethyl CHb),
References and notes
3.56 (td, 2H, J = 6.4 Hz and 1.6 Hz, –SCH2–), 3.79 (t, 2H,
J = 6.8 Hz, HOCH2CH2S–), 3.83–3.89 (m, 4H, benzylic
CHa, HOCH2CH2S– and 50-CHa), 3.95 (d, 1H,
J = 12.0 Hz, benzylic CHb) 3.96 (dd, 1H, J = 10.8 Hz
and 1.6 Hz, 50-CHb), 4.19–4.23 (m, 1H, 40-H), 4.66 (s, 2H,
benzylic CH2), 4.69 (d, 1H, J = 9.2 Hz, 30-H), 6.21 (s, 1H,
anomeric H), 6.67–7.38 (m, 10H, 2· Ph), 8.53 (s, 1H, H-2),
8.61 (s, 1H, H-8); 13C NMR (CD3OD, 100 MHz) d 32.07,
42.31, 61.46,62.34, 69.50, 69.66, 70.57, 74.64, 74.70, 81.51,
82.86, 91.92, 128.77, 128.87, 128.97, 129.00, 129.30,
129.70, 132.19, 138.26, 139.44, 144.64, 149.41, 152.84,
161.74; To a solution of 17b (40.0 mg, 0.065 mmol) in
glacial acetic acid (2.5 mL) was added quickly a warm
solution of mercuric chloride (21.6 mg, 0.08 mmol) in
AcOH (1.3 mL) followed by potassium acetate in acetic
acid (1.4 mL). After heating overnight, the hot solution
was filtered and the precipitate was washed with diethyl
ether. The reaction mixture was evaporated under reduced
pressure and the residue was purified by flash silica gel
column chromatography (CH2Cl2/MeOH = 100:1 ! 30:1)
to give 16 (20.5 mg, 66%) as a colorless solid.
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C.; Carroll, S. S.; Cook, P. D.; Getty, K. L.; MacCoss, M.;
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Altamura, S.; Bhat, B.; Bartholomew, L.; Bosserman, M.
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R.; Eldrup, A. B.; Getty, K. L.; Hou, X. S.; LaFemina, R.
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15. Compound 2a: a white solid; mp 201.8–203.3 ꢁC; MS
(FAB) m/z 298.0 (M+H+); UV (MeOH) kmax 259.5 nm;
25
½aꢀD ꢁ23.81ꢁ (c 0.67, MeOH); 1H NMR (CD3OD,
400 MHz) d 3.24 (d, 1H, J = 11.6 Hz, 20-hydroxymethyl
CHa), 3.47 (d, 1H, J = 11.6 Hz, 20-hydroxymethyl CHb),
3.90 (dd, 1H, J = 12.4 Hz and 2.8 Hz, 50-CHa), 4.05 (dd,
1H, J = 12.4 Hz and 2.0 Hz, 50-CHb), 4.09–4.12 (m, 1H,
40-H), 4.51 (d, 1H, J = 9.2 Hz, 30-H), 6.15 (s, 1H, 10-H),
8.18 (s, 1H, H-2), 8.46 (s, 1H, H-8); 13C NMR (CD3OD,
100 MHz) d 61.35, 63.63, 69.94, 82.20, 84.40, 92.74,
120.29, 142.38, 150.35, 153.45, 157.31. Anal. Calcd for
C11H15N5O5: C, 44.44; H, 5.09; N, 23.56. Found: C, 44.69;
H, 5.48; N, 23.74. Compound 2b: a white solid; mp 147.0–
164.5 ꢁC; MS (FAB) m/z 312.2 (M++1); UV (MeOH) kmax
12. Ho, P.-T. Tetrahedron Lett. 1978, 19, 1623.
13. David, S.; Hanessian, S. Tetrahedron 1985, 41, 643.
14. To a solution of compound 13a (147 mg, 0.30 mmol) in
methanol (10 mL) were added 2-mercaptoethanol
(0.116 mL, 16.5 mmol), and NaOMe (89.1 mg, 16.5 mmol)
and the reaction mixture was heated at reflux for 20 h.
After cooling, the reaction mixture was neutralized with
glacial acetic acid and evaporated. The resulting residue
was purified by flash silica gel column chromatography
(CH2Cl2/MeOH 100:1 ! 30:1) to give 16 (111 mg, 78%) as
a colorless solid with minor formation of thioketal 17b
(40.9 mg, 22%) as a white solid. Compound 16: UV
25
265.0 nm; ½aꢀD ꢁ28.18ꢁ (c 2.02, MeOH); 1H NMR
(CD3OD, 400 MHz) d 3.07 (br s, 3H, N-CH3), 3.17 (d,
1H, J = 12.0 Hz, 20-hydroxymethyl CHa), 3.42 (d, 1H,
J = 12.0 Hz, 20-hydroxymethyl CHb), 3.85 (dd, 1H,
J = 12.8 Hz and 3.6 Hz, 50-CHa), 4.00 (dd, 1H,
J = 12.8 Hz and 1.6 Hz, 50-CHb), 4.04–4.08 (m, 1H, 40-
H), 4.46 (d, 1H, J = 8.8 Hz, 30-H), 6.09 (s, 1H, 10-H), 8.19
(s, 1H, H-2), 8.36 (s, 1H, H-8); 13C NMR (CD3OD,
100 MHz) 49.01, 61.37, 63.73, 70.02, 82.20, 84.42, 92.82,
120.51, 141.72, 150.55, 153.72, 157.56. Anal. Calcd for
C12H17N5O5: C, 46.30; H, 5.50; N, 22.50. Found: C, 46.07;
H, 5.74; N, 22.20. Compound 2c: a white solid; mp 173.5–
199.7 ꢁC; MS (FAB) m/z 298.8 (M+H+); UV (MeOH)
25
1
(MeOH) kmax 250.5 nm; ½aꢀD +3.90ꢁ (c 3.84, MeOH); H
NMR (CD3OD, 400 MHz) d 3.17 (d, 1H, J = 10.0 Hz, 20-
hydroxymethyl CHa), 3.39 (d, 1H, J = 10.0 Hz, 20-hy-
droxymethyl CHb), 3.79 (dd, 1H, J = 11.2 Hz and 3.2 Hz,
50-CHa), 3.90 (dd, J = 11.2 Hz and 2.0 Hz, 50-CHb), 3.98
(s, 2H, benzylic CH2), 4.13–4.16 (m, 1H, 40-H), 4.59 (d,
1H, J = 9.6 Hz, 30-H), 4.61 (s, 2H, benzylic CH2), 6.08 (s,
1H, anomeric H), 6.81–7.34 (m, 10H, 2· Ph), 7.78 (s, 1H,
H-2), 8.32 (s, 1H, H-8); 13C NMR (CD3OD, 100 MHz) d
69.51, 69.86, 70.84, 74.64, 74.79, 81.60, 82.81, 91.99,
125.34, 128.85, 128.95, 129.03, 129.12, 129.32, 129.69,
138.45, 139.46, 146.45, 159.08. Compound 17b: UV
25
kmax 249.5 nm; ½aꢀD ꢁ18.79ꢁ (c 2.29, MeOH); 1H NMR
(CD3OD, 400 MHz) d 3.25 (d, 1H, J = 11.6 Hz, 20-
hydroxymethyl CHa), 3.44 (d, 1H, J = 11.6 Hz, 20-hy-
droxymethyl CHb), 3.84 (dd, 1H, J = 12.4 Hz and
3.2 Hz,50-CHa), 3.98 (dd, 1H, J = 12.4 Hz and 2.4 Hz,
50-CHb), 4.02–4.06 (m, 1H, 40-H), 4.45 (d, 1H, J = 9.2 Hz,
30-H), 6.12 (s, 1H, 10-H), 8.00 (s, 1H, H-2), 8.42 (s, 1H, H-
8); 13C NMR (CD3OD, 100 MHz) d 61.30, 63.48, 69.84,
82.11, 84.37, 92.31, 125.36, 141.56, 146.81, 149.89, 159.22.
Anal. Calcd for C11H14N4O6: C, 44.30; H, 4.73; N, 18.79.
Found: C, 44.14; H, 5.13; N, 18.72.
25
(MeOH) kmax 290.0 nm (sh); ½aꢀD +9.74ꢁ (c 0.72, MeOH);
1H NMR (CD3OD, 400 MHz) d 2.84 (t, 2H, J = 6.4 Hz,
–SCH2–), 3.21 (d, 1H, J = 10.4 Hz, 20-hydroxymethyl