Cytostatic and antiviral 6-arylpurine ribonucleosides. Part 7: Synthesis and evaluation of 6-substituted purine l-ribonucleosides

Article abstract of DOI:10.1016/j.bmcl.2006.07.092

A series of purine l-ribonucleosides 2a-2i bearing diverse C-substituents (alkyl, aryl, hetaryl or hydroxymethyl) in the position 6 were prepared by Pd-catalyzed cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β-l-ribofuranosyl)purine with the

Full text of DOI:10.1016/j.bmcl.2006.07.092

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5290–5293
Cytostatic and antiviral 6-arylpurine ribonucleosides. Part 7:
Synthesis and evaluation of 6-substituted purine L-ribonucleosidesq
a
b
b
b
Michal Hocek,a, Peter Silhar, I-hung Shih, Eric Mabery and Richard Mackman
*
ˇ
´
aGilead Sciences & IOCB Research Center, Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Flemingovo nam. 2, CZ-16610 Prague 6, Czech Republic
bGilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA
Received 15 July 2006; revised 28 July 2006; accepted 31 July 2006
Available online 14 August 2006
Abstract—A series of purine L-ribonucleosides 2a–2i bearing diverse C-substituents (alkyl, aryl, hetaryl or hydroxymethyl) in the
position 6 were prepared by Pd-catalyzed cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-b-L-ribofuranosyl)purine with
the corresponding organometallics followed by deprotection. Unlike their D-ribonucleoside enantiomers that possess strong cyto-
static and anti-HCV activity, the L-ribonucleosides were inactive except for 6-benzylpurine nucleoside 2h showing moderate anti-
HCV effect in replicon assay. A triphosphate of 2h did not inhibit HCV RNA polymerase.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Purine nucleosides 1 bearing C-substituents in position 6
are an important class of biologically active compounds.
6-Methyl- and 6-ethylpurine nucleosides are cytotoxic.1
6-(hydroxymethyl)-,2 6-(fluoromethyl)-,3 6-(difluorom-
ethyl)-4, and 6-(trifluoromethyl)purine5 ribonucleosides
are all strongly cytostatic. 6-Aryl-, 6-hetaryl-, and 6-ben-
zylpurine ribonucleosides were also found6 to possess
significant cytostatic effects. A subclass of them, purine
ribonucleosides bearing 5-membered heterocycles in po-
sition 6, have recently been reported to exert7 strong
anti-HCV activities. Though the mechanism of action
of the latter class of compounds is not yet fully under-
stood, they all inhibit RNA synthesis. In order to
achieve selective inhibition of HCV RNA polymerase,
some additional sugar modifications may be pursued.
From the previous studies it is known that 20- and 50-
deoxyribonucleosides,8 as well as 20-C-methyl-ribonu-
cleosides9 of the 6-aryl- or 6-hetarylpurine series are
inactive, while some carbocyclic homonucleosides still
exert10 cytostatic effects.
L-nucleosides were found12 to possess strong antiviral
activity, lower toxicity and higher metabolic stability
compared to their D-counterparts. Several of them are
clinically used antiviral drugs and some other ones are
in final stages of clinical trials.13 These findings prompt-
ed extensive research of nucleobase- as well as sugar-
modified L-nucleosides in recent years.14
Combining the structural features of the two above-
mentioned classes of biologically active compounds,
we report here on the synthesis and biological activity
of L-ribonucleosides 2 (Fig. 1) derived from purines
bearing diverse C-substituents in position 6. The selec-
tion of substituents was based both on diversity and
on analogy to the most active compounds of the D-ribo
D-Ribonucleosides
R
L-Ribonucleosides
R
Cytostatic:
N
N
R = aryl, hetaryl,
methyl, ethyl,
benzyl
N
N
?
L-Nucleosides, enantiomers of the natural nucleosides,
were first reported11 in the 1960s. Much later some
N
N
N
N
HO
OH
O
O
Anti-HCV:
R = hetaryl
(5-membered)
OH OH
1
OH OH
Keywords: Purines; Nucleosides; Antivirals; HCV.
q
2
Part 6, see Ref. 7.
Corresponding author. Tel.: +420 220183324; fax: +420
220183559; e-mail: hocek@uochb.cas.cz
*
Figure 1. Structures of biologically active purine D-ribonucleosides 1
and the title L-nucleosides 2.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.092

M. Hocek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5290–5293
5291
series: two examples of phenyl groups, three hetaryl,
three alkyl groups and one functionalized, hydroxy-
methyl group.
furyl)purine nucleosides 4d and 4e (analogy to Ref.
6b). Cross-coupling reactions with trimethylaluminum,
triethylaluminum (analogy to Ref. 17), and benzylzinc
chloride (analogy to Ref. 6b) in the presence of
Pd(PPh3)4 in THF at 70 ꢁC gave the corresponding
6-methyl-, 6-ethyl-, and 6-benzylpurines 4f–4h. Cross-
coupling with (benzoyloxymethyl)zinc iodide in presence
of Pd(PPh3)4 in THF at rt gave 6-(benzoyloxymeth-
yl)purine 4i (analogy to Ref. 2). All the acylated nucleo-
sides 4 were deprotected using standard treatment with
NaOMe in methanol to give the series of L-ribonucleo-
sides 2a–2i in good yields.18
All the title L-ribonucleosides 2 were prepared by cross-
coupling reactions15 of the corresponding protected
6-chloropurine nucleoside 3. This intermediate was pre-
pared16 in 61% yield by glycosidation of 6-chloropurine
by commercially available 1,2,3,5-tetra-O-acetyl-b-L-ri-
bose in presence of SnCl4.
The 6-chloropurine nucleoside 3 was then subjected to a
series of cross-coupling reactions with organometallics
(Scheme 1, Table 1). The Suzuki–Miyaura coupling with
phenyl-, 4-methoxyphenyl-, and 3-thienylboronic acid in
toluene catalyzed by Pd(PPh3)4 gave 6-phenyl-, 6-(4-
methoxyphenyl)-, and 6-(3-thienyl)purine nucleosides
4a–4c (analogy to Ref. 6a,7). The Stille reactions with
2-thienyl- and 2-furyl(tributyl)stannanes in DMF cata-
lyzed by PdCl2(PPh3)2 gave 6-(2-thienyl)- and 6-(2-
Title L-nucleosides 2a–2i were subjected to biological
activity screening. In vitro cytostatic activity tests (inhi-
bition of cell growth) were performed using the follow-
ing cell cultures: mouse leukemia L1210 cells (ATCC
CCL 219); human promyelocytic leukemia HL60 cells
(ATCC CCL 240); human cervix carcinoma HeLa S3
cells (ATCC CCL 2.2); and human T lymphoblastoid
CCRF-CEM cell line (ATCC CCL 119). None of these
compounds showed any significant cytostatic effect at
10 lM concentration.
Cl
Cl
N
N
Antiviral activity of L-nucleosides 2a–2i was evaluated
in an HCV subgenomic replicon assay19 and the results
are presented in Table 2. Analogs, 2d and 2h, displayed
antiviral activity with no detectable cytotoxicity in Huh-
7 or MT-4 cells. Close structural analogs of 2d, for
example 3-thienyl (2c) and 2-furyl (2e), were inactive
N
N
N
N
H
N
SnCl4, CH3CN
N
OAc
O
+
OAc
O
3
AcO
OAc
OAc
OAc
OAc
cross-coupling
see, Table 1
Table 2. HCV antiviral activity, cytotoxicity, and HCV NS5B
inhibition
R
R
N
NaOMe
N
N
N
Compound HCV replicona Huh7b
MT-4c
CC50 (lM) CC50 (lM) IC50 (lM)
NS5Bd
N
EC50 (lM)
N
N
N
OH
OAc
2a
2b
2c
2d
2e
2f
>100
>50
>50
56
>50
>100
>50
—
—
—
—
O
O
—
—
—
2a-2h
1,2,4
4a-4h
OH
OAc
OH
OAc
>100
>100
>500
>500
>500
>500
>100
—
—
>100
>500
>500
41
—
—
R
1,2,4
R
a
Ph
CH3
f
2g
2h
2i
—
>500
—
—
>100
—
b
c
4-MeOPh
3-thienyl
2-thienyl
2-furyl
g
CH2CH3
CH2Ph
CH2OH
>500
h
i*
a Antiviral activity in HCV con1 replicon (N = 2).
d
e
*4i, R = CH2OBz
b Cellular toxicity in Huh-7 cells harboring con-1 replicon (N = 2).
c Cellular toxicity in MT-4 cells (N = 2).
d In vitro inhibition toward purified HCV NS5B (N = 2).
Scheme 1. Synthesis of the title L-nucleosides 2.
Table 1. Cross-coupling reactions of 3 with organometallics
Entry
Reagent
Catalyst
Product (yield)
Deprotection (yield)
1
2
3
4
5
6
7
8
9
PhB(OH)2
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
PdCl2(PPh3)2
PdCl2(PPh3)2
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
4a (88%)
4b (85%)
4c (90%)
4d (93%)
4e (92%)
4f (66%)
4g (68%)
4h (62%)
4i (88%)
2a (75%)
2b (87%)
2c (79%)
2d (72%)
2e (76%)
2f (88%)
2g (73%)
2h (62%)
2i (80%)
4-MeOPhB(OH)2
3-ThienylB(OH)2
2-ThienylSnBu3
2-FurylSnBu3
Me3Al
Et3Al
BnZnCl
BzOCH2ZnI

5292
M. Hocek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5290–5293
ˇ´ ´
8. Hocek, M.; Holy´, A.; Votruba, I.; Dvorakova, H. Collect.
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Koh, Y.-h.; Shaw, S. Z.; Zhong, W. Bioorg. Med. Chem.
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indicating that the antiviral structure–activity relation-
ships for 6-modified purine L-ribonucleosides are quite
narrow.
To verify the antiviral mode of action, the triphos-
phate of 2h was prepared20 and the in vitro inhibi-
tion of HCV NS5B polymerase evaluated.21
Inhibition was determined using recombinant NS5B
derived from HCV con1 strain (1b), with a C-termi-
nal hexahistidine tag and the last 21 amino acids
deleted, and a heteropolymeric RNA template.22 No
inhibition of the NS5B polymerase activity was
observed up to 100 lM concentration of inhibitor
(Table 2). In addition, the ability of 2h triphosphate
to serve as a NS5B polymerase substrate in a single-
nucleotide incorporation assay was determined.23 The
incorporation assay was performed using recombinant
NS5B from HCV con1 strain (1b), with a C-terminal
hexahistidine tag and the last 55 amino acids deleted,
a dinucleotide primer (GpC), and a RNA oligonu-
cleotide template (50-CAAAAAAAUGC-30).24 No
incorporation of 2h triphosphate by NS5B was
observed up to concentrations of 1 mM. Further-
more, the triphosphate of L-adenosine was also not
incorporated by NS5B polymerase under the same
conditions. Taken together, these results suggest that
purine L-ribonucleosides are not substrates of the vir-
al polymerase and that the antiviral activity of 2h
was not due to incorporation by NS5B polymerase
into the viral RNA and subsequent termination of
further synthesis, but most likely inhibition of other
cellular or viral targets.
10. Fernandez, F.; Garcia-Mera, X.; Morales, M.; Rodriguez-
Borges, J.; De Clercq, E. Synthesis 2002, 1084.
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11. (a) Smejkal, J.; Sorm, F. Collect. Czech. Chem. Commun.
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Hernandez, B.; Juodawlkis, A.; Tennant, B.; Korba, B.;
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14. Recent examples: (a) Mugnaini, C.; Botta, M.; Coletta,
M.; Corelli, F.; Focher, F.; Marini, S.; Renzulli, M. L.;
Verri, A. Bioorg. Med. Chem. 2003, 11, 357; (b) Zhu, W.;
Chong, Y. H.; Choo, H.; Mathews, J.; Schinazi, R. F.;
Chu, C. K. J. Med. Chem. 2004, 47, 1631; (c) Seela, F.;
Lin, W.; Kazimierczuk, Z.; Rosemeyer, H.; Glacon, V.;
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A.; Zhang, X.; La Colla, P. Nucleosides Nucleotides
Nucleic Acids 2005, 24, 859; (d) Clinch, K.; Evans, G.
B.; Fleet, G. W. J.; Furneaux, R. H.; Johnson, S. W.;
Lenz, D. H.; Mee, S. P. H.; Rands, P. R.; Schramm, V. L.;
Taylor Ringia, E. A.; Tyler, P. C. Org. Biomol. Chem.
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Acknowledgments
This work is a part of the research project Z4 055 0506.
It was supported by the ‘Centre for New Antivirals and
Antineoplastics’ (1M0508), by the Programme of
Targeted Projects of Academy of Sciences of the Czech
Republic (1QS400550501), and by Gilead Sciences, Inc.
(Foster City, CA, USA).
16. Compound 3: a mixture of chloropurine (3.8 g, 20 mmol)
and 1,2,3,5-tetra-O-acetyl-b-L-ribose (6.4 g, 20 mmol) in
acetonitrile (100 ml) was stirred at rt and SnCl4 (5 ml) was
added dropwise. The stirring was continued for 10 h and
the solvent was evaporated. The residue was dissolved in
ethyl acetate (250 ml) and washed with satd aq NaHCO3
(2· 250 ml) and H2O (2· 250 ml). The organic phase was
dried, evaporated, and the residue chromatographed on
silica gel (ethyl acetate) to give 3 as yellowish foam (5 g,
61%), [a]D +15.9 (c 0.35, CHCl3). Spectral data were in
accord with those of D-enantiomer: Buck, I.M.; Reese,
C.B. J. Chem. Soc., Perkin Trans. 1 1990, 2937–2942.
17. (a) Hirota, K.; Kitade, Y.; Kanbe, Y.; Maki, Y. J. Org.
References and notes
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´
Chem. 2005, 3, 3001.
18. Spectral data of compounds 2 and 4 were in accord with
those of the corresponding D-ribonucleosides. Compari-
son of mp and [a]D values of L-nucleosides 2 with those of
D-nucleosides 1: 2a: mp 229–231 ꢁC, [a]D +60.4 (c 0.34,
DMF) [lit.6a for 1a: mp 228–230 ꢁC, [a]D ꢀ56.1 (c 0.5,
DMF)]. Compound 2b: mp 172–176 ꢁC, [a]D +55.9 (c 0.15,
DMF) [lit.6a for 1b: mp 173–175 ꢁC, [a]D ꢀ61.5 (c 0.5,
DMF)]. Compound 2c: mp 198–200 ꢁC, [a]D +60.8 (c 0.39,
DMF) [lit.7 for 1c: mp 196–199 ꢁC, [a]D ꢀ51.8 (c 0.5,
DMF)]. Compound 2d: mp 225–229 ꢁC, [a]D +68.2 (c 0.33,
DMF) [lit.6b for 1d. H2O: mp 126–129 ꢁC, [a]D ꢀ55.3 (c
0.2, DMF)]. Compound 2e: mp 179–181 ꢁC, [a]D +62.5 (c
ˇ
4. Silhar, P.; Pohl, R.; Votruba, I.; Hocek, M. Synthesis
´
2006, 1848.
´
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Tetrahedron 1999, 55, 11109.
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M. Hocek et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5290–5293
5293
0.46, DMF) [lit.6b for 1e. 1/2 H2O: mp 165–168 ꢁC, [a]D
ꢀ54.1 (c 0.2, DMF)]. Compound 2f: mp 206–209 ꢁC, [a]D
+50.6 (c 0.23, DMF) [lit.1 for 1f: mp 209 ꢁC, [a]D ꢀ52.1
(MeOH)]. Compound 2g: mp 192–195 ꢁC, [a]D +48.5 (c
0.20, DMF) [lit.1 for 1g. H2O: mp 105 ꢁC, [a]D ꢀ50
(MeOH)]. Compound 2h: mp 103–106 ꢁC, [a]D +58.6 (c
0.13, DMF) [lit.6b for 1h: mp 97–100 ꢁC, [a]D ꢀ47.7 (c 0.2,
DMF)]. Compound 2i: mp 75–78 ꢁC, [a]D +27.6 (c 0.3,
H2O) [lit.2 for 1i. H2O: mp 65–69 ꢁC, re-measured [a]D
ꢀ27.1 (c 0.3, H2O)].
addition of RNA template and NTP substrates. The
reaction was allowed to proceed for 90 min and then
transferred onto 96-well DE81 filter membranes, washed
three times with 100 mM Na2HPO4, once with ethanol,
and dried. Scintillation fluid was added to the wells and
counts per minute (cpm) were measured in a TopCount.
Radioactivity of each reaction was plotted against the
drug concentration. IC50 was calculated by nonlinear
regression analysis using GraphPad software package.
22. Hung, M.; Gibbs, C. S.; Tsiang, M. Antiviral Res. 2002,
56, 99.
23. Single-nucleotide incorporation assay: the reaction was
carried out in 50 mM HEPES (pH 7.3), 5 mM MgCl2,
10 mM DTT, 20 lM 33P-labeled-GpC primer, 20 lM
oligonucleotide, 1 mM nucleotide triphosphate, and
2 lM of NS5B-delta55 enzyme, at 34 ꢁC for 60 min. The
reaction mixture was resolved on 20% denaturing acryl-
amide gel and quantified by PhosphorImager
(Amersham).
19. Lohmann, V.; Korner, F.; Koch, J.; Herian, U.; Theil-
mann, L.; Bartenschlager, R. Science 1999, 285, 110.
20. Triphosphates were prepared from the corresponding
nucleosides by Chemcyte Inc., San Diego, USA.
21. IC50 determination: The reaction was assembled in 50 mM
Tris/HCl, pH 7.5, 10 mM KCl, 5 mM MgCl2, 1 mM DTT,
1 mM EDTA, 40 ng/ll RNA template, 75 nM NS5B-
delta21 enzyme, 0.5 lCi of [a-33P] adenosine triphosphate,
3 lM ATP, and 500 lM remaining NTP’s. Purified
enzyme was preincubated with compounds for 20 min at
34 ꢁC, after which time the reaction was started by the
24. Cramer, J.; Jaeger, J.; Restle, T. Biochemistry 2006, 45,
3610.