Tricyclic 2′-C-modified nucleosides as potential anti-HCV therapeutics

Article abstract of DOI:10.1021/ol101482h

Promising biological activity in a number of therapeutic areas has been reported for both tricyclic nucleosides and 2′-modified nucleosides. In particular, disubstitution at the C-2′ position of nucleosides has resulted in significant activity against the hepatitis C virus (HCV). Combining this with the observation that tricyclic nucleosides developed in our laboratory have been shown to inhibit the RNA-dependent RNA polymerase NS5B led to the design of a series of 2′-modified tricyclic nucleosides. Details of the synthesis, structural characterization, and preliminary biological results are reported.

Full text of DOI:10.1021/ol101482h

ORGANIC
LETTERS
2010
Vol. 12, No. 20
4466-4469
Tricyclic 2′-C-Modified Nucleosides as
Potential Anti-HCV Therapeutics
Orrette R. Wauchope,† Matthew J. Tomney,† Joseph L. Pepper,† Brent E. Korba,‡
and Katherine L. Seley-Radtke*,†
Department of Chemistry and Biochemistry, UniVersity of Maryland,
Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, and
Department of Microbiology and Immunology, Georgetown UniVersity Medical Center,
3900 ReserVoir Road, N.W., Washington, D.C. 20057
kseley@umbc.edu
Received June 28, 2010
ABSTRACT
Promising biological activity in a number of therapeutic areas has been reported for both tricyclic nucleosides and 2′-modified nucleosides.
In particular, disubstitution at the C-2′ position of nucleosides has resulted in significant activity against the hepatitis C virus (HCV). Combining
this with the observation that tricyclic nucleosides developed in our laboratory have been shown to inhibit the RNA-dependent RNA polymerase
NS5B led to the design of a series of 2′-modified tricyclic nucleosides. Details of the synthesis, structural characterization, and preliminary
biological results are reported.
The hepatitis C virus (HCV) is a blood borne virus that has
affected more than 5 million people in the United States and
nearly 200 million people worldwide.1 HCV is a leading
cause of long-term liver cirrhosis, resulting in liver trans-
plants, liver failure, and, in many cases, death. These
complications are particularly life-threatening for patients that
are coinfected with the human immunodeficiency virus
(HIV). HCV consists of several genotypes, with genotype 1
(HCV-GT1) being the most common. The primary treatment
for HCV-GT1 currently consists of a combination of pegy-
lated interferon administered with the nucleoside analogue
Ribavirin, however the success rate of this treatment is
reported to be less than 50%. Although several promising
nucleoside analogues are currently in clinical trials, most have
failed to make it to market.2,3
The process by which HCV replicates relies on the use of
an RNA-dependent RNA-polymerase (RdRp), in particular,
NS5B. The NS5B polymerase is known as a “right hand”
polymerase made up of a palm (the active site), fingers and
a thumb domain.4 To date, NS5B is the only known site in
which elongation of new virus occurs, designating it as a
prime target for new drug development.
To date a number of 2′-modified nucleosides (Figure
1) have shown potent biological activity against HCV.5,6
Moreover, a thiophene-expanded tricyclic purine nucleoside
(3) Macor, J., E. Annual Reports in Medicinal Chemistry; Macor, J. E.,
Ed.; Elsevier: Oxford, UK, 2009; Vol. 44, Chapter 20, p 398.
(4) Qin, W.; Luo, H.; Nomura, T.; Hayashi, N.; Yamashita, T.;
Murakami, S. J. Biol. Chem. 2002, 227, 2132–2137.
(5) Eldrup, A. B.; Prhavc, M.; Brooks, J.; Bhat, B.; Prakash, T. P.; Song,
Q.; Bera, S.; Bhat, N.; Dande, P.; Cook, P. D.; Bennett, F.; Carroll, S.;
Ball, R. G.; Bosserman, M.; Burlein, C.; Colwell, L. F.; Fay, J. F.; Flores,
O. A.; Getty, K.; LaFemina, R. L.; Leone, J.; MacCoss, M.; McMasters,
D. R.; Tomassini, J. E.; Langen, D. V.; Wolanski, B.; Olsen, D. B. J. Med.
† University of Maryland, Baltimore County.
‡ Georgetown University Medical Center.
(1) Alter, M. J. Gastroenterology 2007, 13, 2436.
(2) Foster, G.; Mathurin, P. AntiViral Ther. 2008, 13, 3
.
Chem. 2004, 47, 5284–5297.
10.1021/ol101482h  2010 American Chemical Society
Published on Web 09/16/2010

Scheme 1. Initial Route to the C-2′-Me Intermediate
Figure 1
properties.
. Modified nucleoside analogues exhibiting anti-HCV
analogue developed in our laboratories has also shown
interesting HCV inhibition (Figure 1).7
In an effort to further explore the potential for increasing
the HCV activity, the tricyclic nucleoside was modified at
the C-2′ position of the sugar (Figure 2). It was hoped that
Intermediate 3 was then treated with trimethylaluminum11
to afford the C-2′ methyl substituted TIPS-protected inter-
mediate, which was then immediately deblocked using TBAF
to provide 4. Trimethylaluminum was used instead of the
more common Grignard and lithium reagents due to reports
of higher yields and the stereoselective addition of the methyl
to the R-face of the sugar.12,13 This approach was desired
since the ultimate goal was to replace the hydroxyl group
(10) 2-(4,5-Diiodo-imidazol-1-yl)-5,5,7,7-tetraisopropyl-1-ꢀ-D-ribofura-
nose (3): Compound 2 (10.0 g, 22.1 mmol), previously dried under high
vacuum, was treated with anhydrous pyridine (200 mL), and the solution
was stirred for 15 min, at which point 1,3-dichloro-1,1,3,3-tetraisopropy-
ldisiloxane (8.5 mL, 26.5 mmol) was added and the reaction was stirred
for 14 h. The solvent was removed under reduced pressure, and the crude
compound was purified by column chromatography eluting with 6:1
hexanes/ethyl acetate to provide 3 as a sticky white foam (12.5 g, 17.3
mmol, 81.4%). The intermediate (9.0 g, 13.0 mmol) was stirred with PCC
(7.0 g, 32.4 mmol) in anhydrous CH2Cl2 (200 mL) over oven 4 Å dried
molecular sieves. The reaction turned from orange to a dark red-brown
color within the first 30 min. The reaction was stirred for 12 h at rt at
which point TLC analysis confirmed the absence of starting material. The
crude reaction mixture was filtered over a pad of celite, and the solvent
was removed under reduced pressure. The crude compound was purified
by column chromatography eluting with 6:1 hexanes/ethyl acetate to provide
Figure 2. Tricyclic C-2′ modified target.
by combining these two leads, a synergistic increase in
inhibition properties against HCV would be realized.
Two synthetic routes were considered in order to realize
the target (Figure 2); adding the methyl group to the
preconstructed tricyclic nucleoside at the last step or
alternatively, adding the methyl group prior to construction
of the tricyclic scaffold. Both routes proceed by way of a
key intermediate (2, Scheme 1), which could be obtained
through a 3-step process using the Vorbru¨ggen8 coupling
method as previously reported from our laboratory.9
The first pathway was quickly abandoned due to the need
for additional protection and deprotection steps and very low
yields. Turning to the second route, intermediate 2 was bis-
protected at the 3′- and 5′-hydroxyls using tetraisoproyld-
isiloxane chloride (TIPDSCl) in preparation for selective
oxidation of the 2′-OH with pyridinium chlorochromate
(PCC) (Scheme 1).10
1
3 as a light-yellow foam (6.0 g, 8.7 mmol, 66.9%). H NMR (CDCl3) δ
0.99 (m, 28H), 3.75 (dd, 1H), 4.00 (dd, 1H), 4.09 (d, 2H), 4.77 (d, 1H),
5.59 (s, 1H), 7.82 (s, 1H). 13C NMR (CDCl3) δ12.3 (multiple peaks), 17.5
(multiple peaks), 60.3, 72.4, 78.7, 84.4, 92.5, 99.2, 139.8, 204.5. HRMS
calculated for C20H34I2N2O5Si2 [MH]+ 693.0175; Found, 693.0175.
(11) 2-(4,5-Diiodo-imidazol-1-yl)-5,5,7,7-tetraisopropyl-3-methyl- 1-ꢀ-
D-ribofuranose (4): Intermediate 3 (4.0 g, 5.8 mmol) was added to anhydrous
CH2Cl2 (100 mL) and cooled to 0 °C. Trimethylaluminum (3 M in hexanes,
6.7 mL, 20.2 mmol) was added, and the mixture was vigorously stirred at
rt for 2 h. The solution was then cooled in an ice bath, quenched with
saturated NaHCO3 solution, extracted with CH2Cl2 (3 × 150 mL), the
organic layers were combined and dried over MgSO4, and the solvent was
removed under reduced pressure. The crude compound was then purified
by column chromatography eluting with 6:1 hexanes/EtOAc to afford 4 as
a white solid (3.0 g, 4.2 mmol, 73.2%). The intermediate (2.0 g, 2.8 mmol)
was dissolved in anhydrous THF (75 mL) and tetrabutylammonium fluoride
(1M in THF, 11.3 mL, 11.3 mmol) was added. The mixture was allowed
to stir at rt for 5 h before the solvent was removed under reduced pressure.
The crude compound was purified by column chromatography eluting with
10:1 EtOAc/MeOH to give 26 as an off white hygroscopic solid (1 g, 2.15
(6) Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish,
P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R. F.; Watanabe, K. A.; Otto,
M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.; Pankiewicz, K. W. J. Med.
Chem. 2005, 48, 5504–5508.
1
(7) Seley-Radtke, K.; Zhang, Z.; Wauchope, O.; Zimmermann, S.;
Ivanov, A.; Korba, B. Nucleic Acids Symp. 2008, 52, 635–636.
(8) Niedballa, U.; Vorbru¨ggen, H. J. Org. Chem. 1974, 39, 3654–3660.
(9) Seley, K. L.; Zhang, L.; Hagos, A.; Quirk, S. J. Org. Chem. 2002,
67, 3365–3373.
mmol, 76.3%). H NMR (CD3OD) δ 1.28 (s, 3H), 3.82 (dd, 1H), 3.95 (d,
2H), 4.08 (d, 1H), 5.67 (s, 1H), 8.12 (s, 1H). 13C NMR (CD3OD) δ 17.6,
61.4, 75.4, 79.4, 81.2, 85.3, 93.8, 96.5, 142.4. HRMS calculated for
C9H12I2N2O4 [MH]+ 466.8966; Found, 466.8966.
(12) Li, N. S.; Piccirilli, J. A. J. Org. Chem. 2006, 71, 4018–4020
.
Org. Lett., Vol. 12, No. 20, 2010
4467

Scheme 2. Synthetic Route to 1
functionality with a fluorine, which is known to proceed
through inversion of configuration by the replacement of the
“up” OH.6
we reported14 a mechanistic study that favors the preferential
formation of the guanosine analogue when the sugar is a
ribose,9 however this did not prove fruitful with the C-2′-
modification and the xanthosine was the major product in
all cases.
While the use of trimethylaluminum was reported to give
stereoselective yields, to our knowledge, no spectroscopic
proof for this selective addition has been reported. In an effort
to confirm the correct isomer was obtained, a series of 1D-
NOE NMR experiments were run. Irradiations of the
anomeric proton, 2′-Me, 3′-H, and the 4′-H showed similar
correlations thus providing insight as to the stereochemistry
of the sugar. Specifically, the anomeric proton was found to
have the strongest correlation to the 2′-Me group. This signal
was normalized to 1 as a reference for measuring the strength
of other correlations. The anomeric proton showed a relative
abundance of 100% (normalized), the 4′-H showed a relative
abundance of 38%, while the 3′-H showed relatively no
correlation to the 2′-Me (less than 1%). These correlations
strongly suggest that the Me addition occurred to the R-face
of the C-2′-position as expected.
Proceeding with the construction of the tricyclic base,
blocking the hydroxyl groups was undertaken next. Employ-
ing the benzyl (Bn) group, the protected intermediate was
obtained in a 65% yield as shown in Scheme 2. The
remaining steps proceeded as previously reported for the
guanosine ribose analogue.9 Replacement of the C-5 iodo
with the formyl group followed by conversion to the oxime,
which was then dehydrated to give the cyano modified 5.
Derivitization of C-4 with thioglycolamide then afforded
an intermediate which was then treated with base to give
the bicyclic analogue 6. Closure of the third ring with
standard conditions is known to lead to a competitive
formation of the desired tricyclic guanosine analogue (1) and
the undesired tricyclic xanthosine analogue (8), a problem
also encountered with the formation of guanosine. Previously
(13) Li, N. S.; Lu, J.; Piccirilli, J. A. J. Org. Chem. 2009, 74, 2227–
2230.
(14) Zhang, Z.; Wauchope, O.; Seley, K. Tetrahedron 2008, 64, 10791–
10797.
(15) New procedure for 1-[(5-Aminoimidazo[4′,5′:4,5]thieno[3,2-d]py-
rimidin-3-yl-7-one)]-ꢀ-D-ribofuranose 2,3,5-Triol (1) and 1-[(5-hydroxy-
limidazo[4′,5′:4,5]thieno[3,2-d]pyrimidin-3-yl-7-one)]-ꢀ-D-ribofuranose (8):
In a steel reaction vessel, compound 6 (2.62 g, 4.38 mmol) was dissolved
in 40 mL anhydrous MeOH, NaOH (1.76 g, 44 mmol) was added, and the
mixture was stirred at room temperature for 30 min until a clear solution
was obtained. CS2 (1.6 mL, 26.3 mmol) was added, the vessel was sealed,
and the reaction was heated in an oil bath at 145 °C for 18 h. The vessel
was cooled, and the solvent was removed under reduced pressure to give
an orange solid to which THF (80 mL) was added. The mixture was cooled
to 0 °C and 11.4 mL tert-butyl hydroperoxide (5 M in decane) was added.
This mixture was stirred at 0 °C for 4 h until TLC analysis confirmed the
disappearance of the starting material. The reaction mixture was then
transferred to a steel reaction vessel, cooled to -78 °C, and NH3 was
bubbled into the solution for 15 min. The reaction vessel was sealed and
heated in an oil bath at 120 °C for 18 h. The vessel was cooled, and the
reaction mixture was evaporated to dryness under vacuum to give a yellow
solid. The crude mixture was purified by column chromatography eluting
with hexanes/EtOAc (3:1) to afford the guanosine and xanthosine analogues
as colorless oils. The crude compounds were then dried before anhydrous
CH2Cl2 (15 mL) was added. At this point, 12 molar equiv of BF3•Et2O and
12 molar equiv of EtSH were added to the solution dropwise. The reaction
was allowed to proceed for 48 h at room temperature before TLC analysis
confirmed complete product formation. The solvent was removed under
reduced pressure to give a crude solid. The crude compound was purified
via column chromatography using a solvent system of 10:1 MeOH/EtOAc.
This provided the guanosine analogue 1 and the xanthosine analogue 8 in
a 1.2:1 ratio. Each final compound was rinsed 3 times with CH2Cl2, rinsed
3 times with EtOAc, and finally rinsed 3 times with a 5:1 mixture of EtOAc/
1
MeOH. Guanosine 1: H NMR (DMSO-d6) δ 1.44 (s, 3H), 3.65 (dd, 1H),
4.20 (d, 2H), 4.39 (d, 1H), 5.43 (broad m, 3H), 5.72 (s, 1H), 6.51 (s, 2H),
1
8.35 (s, 1H), 10.98 (broad s, 1H). Xanthosine 8: H NMR (DMSO-d6) δ
1.12 (s, 3H), 3.82-3.93 (m, 1H), 4.01-4.11 (m, 1H), 5.40 (broad m, 2H),
5.83 (s, 1H), 8.45 (s, 1H), 10.68 (s, 1H), 11.42 (s, 1H).
4468
Org. Lett., Vol. 12, No. 20, 2010

In examining the reaction more closely, it appeared that
the water present in the 30% aqueous hydrogen peroxide
solution used was displacing the intermediate sulfenic (SOH)
or sulfinic (SO2H) acid group on 7 (Scheme 2) more rapidly
than the ammonia, thereby resulting in Bn-protected xan-
thosine 8 as the major product. In an effort to overcome this
obstacle, use of a non-nucleophilic peroxide was tried and,
indeed, proved highly successful. Thus, by substituting tert-
butyl hydrogen peroxide in the ring closure reaction, the ratio
of the tricyclic guanosine to xanthosine was increased
significantly from 1:20 to 1.2:1.15 Deblocking as before
provided 1 and 8, with the desired guanosine 1 being the
major product.
tested against other viruses as soon as sufficient amounts
can be obtained.
In summary, two novel tricyclic C-2′ modified nucleosides
were successfully obtained and screened for potential activity
against HCV. Although the compounds did not show activity,
possibly due to their poor solubility in the assay system, the
new ring closure protocol significantly increased the ratio
of the desired guanosine analogue so that scaleup can now
be accomplished, however it still appears that more anhy-
drous conditions may be necessary in order to completely
eliminate the formation of the xanthosine side product. Future
efforts are focused on pursuit of the analogous 2′-fluoro, 2′-
methyl compounds. The results of those studies will be
reported elsewhere as they become available.
Compounds 1 and 8 were then tested for anti-HCV activity
(genotypes 1a and 1b) and cytotoxicity in two separate HCV
replicon-containing cell lines using an established16 standard-
ized three day assay. Unfortunately, neither compound
showed any significant activity at concentrations up to 10
µM. It should also be noted that neither compound exhibited
cytotoxicity at concentrations of 100 µM. While initial results
against HCV were disappointing, the compounds will be
Acknowledgment. This work was generously supported
by the National Institutes of Health (R01 GM076345, KSR).
The anti-HCV assays were supported by NIAID contract
N01-AI-30046 to GUMC (BEK). We are thankful for this
support.
Supporting Information Available: Experimental details,
1H, 13C NMR and HRMS data provided. This material is
available free of charge via the Internet at http://pubs.acs.org.
(16) Korba, B. E.; Montero, A. B.; Farrar, K.; Gaye, K.; Mukerjee, S.;
Ayers, M.; Rossignol., J.-F. AntiVir. Res. 2007, 77, 56–63.
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