Synthesis and anti-hepatitis C virus activity of nucleoside derivatives of N3,5′-anhydro-4-(β-D ribofuranosyl)-8-azapurin-2-ones

Article abstract of DOI:10.1081/NCN-200059319

A series of N³,5′-Anhydro-4-(β-D-ribofuranosyl)-8- azapurin-2-ones were prepared in multistep reactions from uridine as potential anti-hepatitis C virus (HCV) agents. The synthetic details as well as biological evaluations are discussed. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200059319

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SYNTHESIS AND ANTI-HEPATITIS C VIRUS ACTIVITY OF
NUCLEOSIDE DERIVATIVES OF N 3,5′-ANHYDRO-4-(β-D-
RIBOFURANOSYL)-8-AZAPURIN-2-ONES
Abdalla E. A. Hassan a , Peiyuan Wang a , Tamara R. McBrayer b , Phillip M. Tharnish b
,
Lieven J. Stuyver b , Raymond F. Schinazi c , Michael J. Otto b & Kyoichi A. Watanabe a
a Department of Chemical Sciences , Pharmasset, Inc. , Tucker, Georgia, USA
b Department of Biological Sciences , Pharmasset, Inc. , Tucker, Georgia, USA
c Department of Pediatrics , Emory University, School of Medicine/Veterans Affairs Medical
Center , Decatur, Georgia, USA
Published online: 15 Nov 2011.
To cite this article: Abdalla E. A. Hassan , Peiyuan Wang , Tamara R. McBrayer , Phillip M. Tharnish , Lieven J. Stuyver ,
Raymond F. Schinazi , Michael J. Otto & Kyoichi A. Watanabe (2005) SYNTHESIS AND ANTI-HEPATITIS C VIRUS ACTIVITY OF
NUCLEOSIDE DERIVATIVES OF N 3,5′-ANHYDRO-4-(β-D-RIBOFURANOSYL)-8-AZAPURIN-2-ONES, Nucleosides, Nucleotides and
Nucleic Acids, 24:5-7, 961-964, DOI: 10.1081/NCN-200059319
To link to this article: http://dx.doi.org/10.1081/NCN-200059319
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5--7):961–964, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059319
SYNTHESIS AND ANTI-HEPATITIS C VIRUS ACTIVITY OF NUCLEOSIDE
DERIVATIVES OF N3,5’-ANHYDRO-4-(b-D-RIBOFURANOSYL)-8-AZA-
PURIN-2-ONES
5
Abdalla E. A. Hassan and Peiyuan Wang
Department of Chemical Sciences,
Pharmasset, Inc., Tucker, Georgia, USA
5
Tamara R. McBrayer, Phillip M. Tharnish, and Lieven J. Stuyver
Department
of Biological Sciences, Pharmasset, Inc., Tucker, Georgia, USA
5
Raymond F. Schinazi
Department of Pediatrics, Emory University, School of Medicine/
Veterans Affairs Medical Center, Decatur, Georgia, USA
5
Michael J. Otto
Department of Biological Sciences, Pharmasset, Inc., Tucker, Georgia,
USA
5
Kyoichi A. Watanabe
Department of Chemical Sciences, Pharmasset, Inc., Tucker,
Georgia, USA
A series of N3,5’-Anhydro-4-(b-D-ribofuranosyl)-8-azapurin-2-ones were prepared in multistep
reactions from uridine as potential anti-hepatitis C virus (HCV) agents. The synthetic details as well as
biological evaluations are discussed.
5
INTRODUCTION
Hepatitis C virus (HCV) has chronically infected an estimated 170 million
people around the world. HCV infection is the major cause of chronic liver disease,
often leading to hepatic failure and hepatocellular carcinoma.[1] The currently
approved therapy for chronic hepatitis C is long term treatment with interferon-a
(INF-a), either alone or in combination with ribavirin.[2] However, the low rate of
response and the associated adverse effects of such treatments, along with the lack
Supported in part by NIH grants 1R43 AI-52868 (biology) and 1 R43 AI-056720 (chemistry). Dr. Schinazi is a
founder and consultant of Pharmasset, Inc. His laboratory did not receive any funding from Pharmasset, Inc.
Parts of this work were presented at the International Society for Nucleosides, Nucleotides & Nucleic Acids,
XVI International Roundtable, Minneapolis, Minnesota, USA, September 12--16, 2004.
Address correspondence to Peiyuan Wang, Department of Chemical Sciences, Pharmasset, Inc., 1860
Montreal Road, Tucker, GA 30084, USA.
961
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962
A. E. A. Hassen et al.
FIGURE 1
of preventive vaccine necessitates the search for new therapies.[2,3] During the
course of our studies to find small molecules that selectively inhibit HCV, we
discovered that N9,5’-anhydro-4-(b-D-ribofuranosyl)-v-triazolo[4,5-b]pyridin-2-one (1)
inhibits the production of HCV-RNA, in the HCV-subgenomic replicon cell line
(Huh7 cells) EC50 = 19.7 mM and EC90 = 79.8 mM.[4,5] Using 1 as a lead, we
synthesized the hypoxanthine analogue 2 (Figure 1) and several 8-azapurine
derivatives (7a--d, 10) as a part of our efforts in search of superior agents. Four
compounds (2, 7a, 7b and 10) emerged from this investigation as more potent
anti-HCV agents (Table 1). However, this increase in antiviral activity was
accompanied by increased cytotoxicity.
RESULTS AND DISCUSSION
Chemistry
Uridine was converted to 5-bromo-2’,3’-O-isopropylidene-5’-O-mesyl derivative
3a,[6] followed by NaN3 treatment in DMF affording the 5’-azido-5’-deoxy
derivative 3b. Upon heating 3b in DMF at 110--120°C for 30 h, the [2,3]-dipolar
addition of the 5’-azido group to the 5,6-double bond occurred with concomitant
elimination of HBr from the adduct resulting in the formation of 9,5’-cyclo-
nucleoside 4 (Scheme 1).
TABLE 1 Anti-HCV Activity of Synthesized Compounds in the Huh 7
Replicon Cells
Compounds
EC90 (mM)
CC50 (mM)
1
2
79.8
23.7
10.9
<6.25
>100
>100
>100
43.2
30.6
24.4
11.0
6.7
>100
>100
>100
32
7a
7b
7c
7d
8
10

A. E. A. Hassen et al.
963
Treatment of 4 with Ph3P, imidazole, iPr2NEt and I2 in toluene at 95--100°C
gave 6-imidazolyl derivative 5 in 81% yield. When 5 was treated with NH4OH for
24 h at room temperature, the 8-aza-isoguanine derivative 6a was obtained. Various
6-(substituted amino) derivatives 6b--d were prepared directly from 4 by treatment
with triisopropylbenzenesulfonyl chloride and 4-(dimethylamino)pyridine, followed
by reaction with the corresponding amines. Compounds 6a--d were deblocked to
the diols (7a--d) by acid hydrolysis. Acid hydrolysis of 4 yielded the 8-aza-
xanthine analogue 2. Cyanomethylation of 4 with BrCH2CN, NaH and LiBr in
DMF, followed by acid hydrolysis afforded 8. Thiation of 4 with Lawesson’s
reagent in ClCH2CH2Cl at reflux gave 9, which was hydrolyzed to give 9,5’-
anhydro-(3-b-D-ribofuranosyl)-8-aza-6-thioxanthine 10.
Anti-HCV Subgenomic Replicon Assay
HCV-replicon RNA-containing Huh7 cells (Clone A cells; Apath, LLC, St.
Louis, MO) were kept in exponential growth in DMEM media (high glucose, no
pyruvate) containing 10% fetal bovine serum, 1X non-essential amino acids,
penicillin-streptomycin-glutamine (100 units/mL, 100 mg/mL, and 2.92 mg/L,
respectively), and G418 (500 to 1000 mg/mL).[7] Antiviral assays were performed
in the same media without G418. Cells were seeded in a 96-well plate at 1000 cells
per well and test compounds were added immediately after seeding. At the end of
SCHEME 1 Reagent: (a) NaN3/DMF, 80°C; (b) DMF, 110--120°C, 30 h; (c) Ph3P/imidazole/iPr2NEt/I2/toluene;
(d) Et3N/DMAP/TIPSCI/amine; (e) 1NHCl/THF, 90°C; (f) 60% NaH/LiBr/BrCH2CN/DMF; (g) Lawesson’s reagent/
DCE, reflux; (h) CF3CO2H/H2O, 50--55°C.

964
A. E. A. Hassen et al.
the 4-day incubation, total cellular RNA was isolated (RNeasy 96 kit, Qiagen, CA).
Replicon RNA and the internal control (TaqMan Ribosomal RNA control
Reagents, Applied Biosystems, CA) were amplified in a single-step multiplex RT-
PCR protocol, as recommended by the manufacturer. The HCV primers and
probe were designed using the Primer Express software (Applied Biosystems, CA)
and cover highly conserved 5’ untranslated region sequences. The results are
summarized in Table 1.
CONCLUSIONS
It is apparent from the biological results that a substituent on C-6 appears to
contribute to the toxicity of these compounds. Among C-6-amino substituent, the
most active one was the methylamino analogue, but it was also toxic. It seems that
the activity would be reduced as the size of substituent on the amino group grows
larger. Cyanomethyl substitution on C-7 did not show significant anti-HCV activity.
The 6-thio analogue exhibited moderate anti-HCV activity and toxicity.
When tested in cell-free systems, this class of compounds did not inhibit
purified NS5B RNA-dependent RNA polymerase in vitro. Studies on the
mechanism(s) of action of these and related compounds are underway. Since the
mode of action this class of compounds is different from the known NS5B
inhibitors, identification of compounds demonstrating a better anti-HCV activity to
cytotoxicity profile would be of interest.
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