Synthesis and antiviral activity of cyclopropyl-spirocarbocyclic adenosine, (4R,5S,6R,7R)-4-(6-amino-9H-purin-9-yl)-7-(hydroxymethyl)spiro[2.4]heptane-5,6- diol against hepatitis C virus

Article abstract of DOI:10.1016/j.bmcl.2011.05.012

An efficient method was developed for the synthesis of 6-exocyclic methylene carbocyclic intermediate 4. The Simmons-Smith cyclopropanation protocol was applied on the 6-exocyclic methylene of intermediate 4 and demonstrated its utility for the synthesis of novel class of a spiro-carbocyclic nucleoside analog 8. The titled compound 8 demonstrated a significant antiviral activity against HCV with EC₅₀ values of 0.273 and 0.368 μM in genotypes 1A and 1B, respectively.

Full text of DOI:10.1016/j.bmcl.2011.05.012

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3982–3985
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiviral activity of cyclopropyl-spirocarbocyclic adenosine,
(4R,5S,6R,7R)-4-(6-amino-9H-purin-9-yl)-7-(hydroxymethyl)spiro[2.4]heptane-
5,6-diol against hepatitis C virus
Srinivas Gadthula a, Ravindra K. Rawal a, Ashoke Sharon a, Dong Wu a, Brent Korba b, Chung K. Chu a,
⇑
a Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA 30602, USA
b Georgetown University Medical Center, Rockville, MD 20850, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient method was developed for the synthesis of 6-exocyclic methylene carbocyclic intermediate 4.
The Simmons–Smith cyclopropanation protocol was applied on the 6-exocyclic methylene of intermedi-
ate 4 and demonstrated its utility for the synthesis of novel class of a spiro-carbocyclic nucleoside analog
8. The titled compound 8 demonstrated a significant antiviral activity against HCV with EC50 values of
Received 11 March 2011
Revised 2 May 2011
Accepted 3 May 2011
Available online 11 May 2011
0.273 and 0.368 lM in genotypes 1A and 1B, respectively.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Carbocyclic nucleoside
Anti-HCV
NS5B
X-ray structure
Hepatitis C virus (HCV) belongs to flaviviridae family.1 It is one
of the most clinically important infection affecting liver with high
morbidity and mortality worldwide.2 The world health organiza-
tion estimates that chronic infection with HCV occurs in approxi-
mately 180 million of the world population and 3–4 million new
infection occurs each year.3 According to the Centers for Disease
Control and Prevention (CDC) over 3 million people have chronic
HCV infection in the US.
HCV is an RNA virus which possesses a single-stranded posi-
tive-sense RNA as the viral genome.4 This viral RNA plays impor-
tant roles during viral replication, as it serves as an mRNA for
viral protein synthesis, a template for RNA replication as well as
a nascent RNA genome for a newly formed virus.5 There is no
anti-HCV vaccine available today. Because of the significant unde-
sirable side effects of the current combination therapy of subcu-
effective antiviral therapy against hepatitis C should encompass
a broad spectrum of activity against all HCV genotypes; shorten
treatment duration, minimal side effects and a high barrier to
resistance. The HCV non-structural protein NS5B RNA-dependent
RNA polymerase is a key component of the replicative complex
and is responsible for initiating and catalyzing viral RNA synthe-
sis.3,8 Therefore, the HCV NS5B is an attractive target for the cur-
rent drug discovery and development of anti-HCV agents. There
are two major subclasses of NS5B inhibitors: nucleoside analogs,
which are anabolized to their active triphosphates, which act as
alternative substrates for the polymerase, and non-nucleoside
inhibitors (NNIs), which bind to allosteric regions on the protein.
Nucleoside or nucleotide inhibitors mimic natural polymerase
substrate and act as chain terminators. They inhibit the initiation
of RNA transcription and elongation of a nascent RNA chain.
Several NS5B nucleoside/nucleotide polymerase inhibitors have
been in clinical trials as shown in Figure 1.9–13 20-C-Methylcytidine
(NM107), the valine ester of 20-C-methylcytidine(valocitabine,
NM283),10 was the first polymerase inhibitors in clinical trials,
which was discontinued due to the GI toxicity. The second nucleo-
side inhibitor, 40-C-azido-nucleoside (R1479) as its tri-isobutyl es-
ter prodrug (R1626), have been developed by Roche,11 however, it
was discontinued due to the haematopoetic toxicity. Currently,
Roche and Pharmasset are developing R7128 (mericitabine), a pro-
taneous pegylated INF-a and the ribavirin, there is an urgent
unmet medical need to develop anti-HCV therapies that are effec-
tive, well-tolerated and safe for the treatment of chronic HCV
infection.6 The direct-acting antivirals (DAAs) are the compounds
that target a specific viral protein. Presently, four major classes of
DAAs are being investigated in phase II/III clinical trials: inhibi-
tors of NS3 protease, allosteric NS5B polymerase, and NS5B poly-
merase.6,7 The major challenges for these DAAs include safety,
pan-genotypic activity, and/or emergence of resistant strains. An
drug of b-D a
-20-deoxy-20- -fluoro-20-C-methylcytidine (PSI6130).12
Idenix has been developing a purine analogue, 20-C-methylguano-
sine monophosphate prodrug (IDX184),13 however, it is currently
⇑
Corresponding author. Tel.: +1 706 542 5379; fax: +1 706 542 5381.
E-mail address: DCHU@rx.uga.edu (C.K. Chu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.012

S. Gadthula et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3982–3985
3983
Figure 1. Nucleoside/ nucleotide polymerase inhibitors in clinical trials.
Scheme 1. Synthesis of compound 8. Reagents and conditions: (a) Refs. 17 and 18; 44% yield; (b) (i) LDA/THF, ꢀ78 °C, 3 h; then Eschenmoser’s salt, ꢀ78 °C, 3 h, rt, 8 h; (ii)
CH3I, 4 h, rt, 10% NaHCO3, 83% yield; (c) NaBH4/CeCl3ꢁ7H2O/MeOH, ꢀ78 °C to 0 °C, 90% yield; (d) Et2Zn/CH2I2, ether, reflux, 93% yield; (e) 6-chloropurine, Ph3P/DIAD, THF, rt,
26.6%; (f) NH3/MeOH, 100 °C, two steps 48% yield; (g) TFA/H2O, 80% yield.
under clinical hold due to the potential liver toxicity concern by
the FDA. A uridine analogue in a prodrug (PSI7977)9 form can po-
tently inhibit HCV replication and is considered a second genera-
tion of nucleoside inhibitor. Recently, a 30,50-cyclic phosphate
analogue, PSI-938, has also been reported as a potent anti-HCV
agent.6
ring. As a consequence, the glycosidic bond is resistant to nucleoside
phosphorylase as well as nucleoside hydrolase, which renders the
carbocyclic nucleosides more stable towards metabolic degrada-
tion.14 Due to these features, carbocyclic nucleosides have received
much attention as potential chemotherapeutic agents.15,16 Carbovir
and entecavir are examples of the result of these efforts. As a part of
our ongoing antiviral drug discovery program, a number of carbocy-
clic nucleoside analogues have been synthesized and evaluated for
Carbocyclic nucleoside is an interesting class of compounds in
which a methylene group replaces the oxygen atom of a furanose

3984
S. Gadthula et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3982–3985
Table 1
their potential antiviral activity against HCV, and an interesting
novel nucleoside was discovered. Herein, we report the synthesis
and anti-HCV activity of hitherto unreported (4R,5S,6 R,7R)-4-(6-
amino-9H-purin-9-yl)-7-(hydroxymethyl)spiro[2.4]heptane-5,6-
diol (8).
Pseudorotational angle and conformational properties
Compound 8
c
mmax (Puckering
P (Pseudorotational
angle)
(Gamma) amplitude)
Computational ꢀ65.7
X-ray
ꢀ71.0
38.0
44.8
187.6
173.1
In order to synthesize the target nucleoside 8 (Scheme 1), a
ketone 3 was used as the key intermediate, which was synthe-
sized from
D-ribose in nine steps. D-Ribose was converted to
the truncated Newton (TCNG) method using OPLS_2001 force field
with the GB/SA continuum water solvation model26 followed by
Macromodel energy minimization27 using the OPLS_2005 force
field. The lowest energy conformer was used to estimate the
pseudorotational angle and conformational properties. The struc-
ture of compound 8 was further confirmed by the single-crystal
X-ray diffraction study28 (CCDC 816703) and the ORTEP diagram
is shown in Figure 2.29 The conformational properties obtained
by the empirical calculation, as well as by the crystal structure re-
vealed that the nucleoside carbocyclic ring adopts the 20-endo
(south conformation) with anti-base disposition. The lowest en-
ergy conformation found was superposed with the crystallograph-
ically determined structure with respect to the carbocyclic sugar
ring atoms. The RMSD for the spiro-carbocyclic sugar ring atom
positions was 0.058 Å, and the pseudorotational parameters
compound 2 using reported methods.17,18 The exocyclic methy-
lene group was introduced at the 6-position by quenching the
lithium enolate of the ketone 2 with the Eshenmoser’s salt19
(Mannich base), followed by the Hoffmann degradation
method,20 provided an enone 3 in 83% yield. This process is
quite convenient and can be generally applied to the chemistry
of carbocyclic nucleoside. The resultant enone 3 was treated
with sodium borohydride in the presence of cerium(III) chloride
heptahydrate, which exclusively provided the allylic alcohol key
intermediate 421,22 in 90% yield. For the synthesis of the novel
spiro nucleoside, several methods have been tried, however,
the Simmon–Smith cyclopropanation23 of the carbocyclic inter-
mediate 4 using diethyl zinc and diiodomethane under reflux
conditions in diethyl ether was found to produce the best results
and gave the key spiro-intermediate 522 in 93% yield. The spiro
alcohol 5 was coupled with 6-chloropurine under Mitsunobu
conditions, which gave an inseparable mixture of compound 6
and the by-product, reduced diisopropylazodicarboxylate (DIAD).
The mixture was treated with saturated methanolic ammonia in
a steel bomb at 80 °C to give compound 7 in low yield (ꢂ48%).
The low coupling yield is due to the formation of several uniden-
tified by-products. Finally, the compound 7 was deprotected
using a TFA/H2O (2:1, v/v) solution at 50 °C to give the target
nucleoside 8 in 80% yield.24
pseudorotational angle (P), dihedral angle of O50–C50–C40–C30 (
and puckering amplitude ( max) were in close agreement (crystal:
max = 44.8; lowest energy conformer:
max = 38.0). The orientation of hydroxyl
c)
m
P = 173.1,
P = 187.6,
c
= ꢀ71.0,
m
c
= ꢀ65.7,
m
groups and the adenine base was also in between the modeled
and experimentally determined structures (Fig. 3).
The antiviral activity and cytotoxicity of the synthesized com-
pound 8 was evaluated in the HCV sub-genomic RNA replicon as-
say system in Huh7 ET cells as previously described.30 The
results are summarized in comparison to the positive control, 20-
C-Me-cytosine (20-C-Me-C)31 as shown in Table 2. As the modifica-
tions of the sugar portion of 20-C-methyladenosine (20-C-Me-A)
with the cyclopropyl-spirocarbocyclic sugar exhibited significant
Since the conformation of nucleoside/nucleotide analogues
plays an important role in their biological activity as well as to
understand the conformation of the novel class of the spiro nucle-
oside,25 the conformation of the nucleoside 8 was studied. The con-
formational properties were studied using
empirical calculation as well as X-ray crystallography (Table 1).
anti-HCV activity with EC50 values of 0.273 and 0.368
type A and B, respectively.
lM in geno-
a computational
The synthesized cyclopropyl-spirocarbocyclic nucleoside 8 was
tested against cowpox, vaccinia, HBV, HSV-1 & 2, VZV, SARS, Flu
The conformational studies of compound 8 were performed by
Figure 2. ORTEP diagram showing displacement ellipsoid plot of the X-ray crystal structure of 8.

S. Gadthula et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3982–3985
3985
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.012.
References and notes
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8. Ishii, K.; Tanaka, Y.; Yap, C.; Aizaki, H.; Matsuura, Y.; Miyamura, T. Hepatology
1999, 29, 1227.
9. Brown, N. A. Expert Opin. Invest. Drugs 2009, 18, 709.
10. Pierra, C.; Benzaria, S.; Amador, A.; Moussa, A.; Mathieu, S.; Storer, R.; Gosselin,
G. Nucleosides, Nucleotides Nucleic Acids 2005, 24, 767.
11. Pockros, P.; Nelson, D.; Godofsky, E.; Rodriguez Torres, M.; Everson, G.; Fried,
M.; Ghalib, R.; Harrison, S.; Nyberg, L.; Shiffman, M. Hepatology 2008, 48, 385.
12. Stuyver, L.; McBrayer, T.; Tharnish, P.; Clark, J.; Hollecker, L.; Lostia, S.;
Nachman, T.; Grier, J.; Bennett, M.; Xie, M. Antiviral Chem. Chemother. 2006, 17,
79.
Figure 3. Stereoview of the crystallographically determined structure 8 (green) and
its lowest energy conformation (gray) by computational empirical calculation.
13. Zhou, X.-J.; Pietropaolo, K.; Chen, J.; Khan, S.; Sullivan-Bolyai, J.; Mayers, D.
Antimicrob. Agents Chemother. 2011, 55, 76.
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17. Jin, Y. H.; Liu, P.; Wang, J.; Baker, R.; Huggins, J.; Chu, C. K. J. Org. Chem. 2003, 68,
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18. Moon, H. R.; Choi, W. J.; Kim, H. O.; Jeong, L. S. Tetrahedron: Asymmetry 2002,
13, 1189.
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Engl. 1971, 10, 330.
20. Hofmann, A. W. Ber 1881, 14, 2725.
21. Shuto, S.; Obara, T.; Toriya, M.; Hosoya, M.; Snoeck, R.; Andrei, G.; Balzarini, J.;
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Table 2
In vitro anti-HCV activity and cytotoxicity of compound 8 in the HCV RNA replicon
Huh7 assay in comparison to the known 20-C-methylcytidine (20-C-Me-C)
SIc
a
a
b
Compound
Genotype
EC50
(
l
M)
EC90
(
l
M)
CC50 (lM)
Compound 8
1A
1B
0.273
0.368
3.0
1.2
>100
>100
>366
>272
20-C-Me-C
1B
1.7
8.2
>300
>167
a
b
c
Effective concentration required for reducing HCV level by 50% and 90% in
5 days.
Cytotoxicity concentration required for reducing the rRNA levels by 50% in
5 days.
Selectivity index (SI) = CC50/EC50
22. Gaudino, J. J.; Wilcox, C. S. J. Am. Chem. Soc. 1990, 112, 4374.
23. Simmons, H. E.; Smith, R. D. J. Am. Chem. Soc. 1959, 81, 4256.
24. (4R,5S,6R,7R)-4-(6-Amino-9H-purin-9-yl)-7-(hydroxymethyl)spiro[2.4]heptane-
5,6-diol (8). Compound 7 (100 mg, 0.258 mmol) was dissolved in CF3CO2H/H2O
(2:1, v/v) (50 mL) and heated to 50 °C for 3 h. The solvent was removed under the
vacuum and the residue was co-evaporated with ethanol (3 ꢃ 20 mL) under
vacuum. The resultant residue was purified by combiflash chromatography (12%
.
Table 3
Antiviral activity of compound 8 when tested against various viruses in cell culture
MeOH/CH2Cl2) to afford 8 (60 mg, 80%) as a white foam. Mp 120–122 °C. ½a D24
ꢄ
a
b
Virus
Assay
EC50
(l
M)
CC50 (lM)
SIc
5.01 (c 0.45, CHCl3); UV(MeOH) kmax 261 nm (e 12885, pH 2), 261 nm (e 13844,
Vaccinia
Measles
CPE
NR
Visual
NR
Visual
42.7
>300
49
15
13
10
>7.1
5
2
4
3
pH 7), 261 nm (e
14038, pH 11); 1H NMR (500 MHz, DMSO-d6) d 8.71 (s, 1 H, 2-
H), 8.58 (s, 1 H, 8-H), 7.73 (br s, 2H, NH2), 5.58 (d, J = 6 Hz, 1H, 10-H), 5.24–5.30
(m, 3H, 20-H, 20& 30-OH, D2O exchange), 5.03–5.07 (m, 1H, 50-OH, D2O exchange),
4.53 (d, J = 2.5 Hz, 1H, 30-H), 3.91–3.95 (m, 2H, 50-H), 2.41–2.43 (m, 1H, 40-H),
1.12–1.16 (m, 1H, -cp), 1.02–1.09 (m, 2 H, -cp), 0.00–0.04 (m, 1 H, -cp); 13C NMR
(125 MHz, DMSO-d6) d 155.9, 151.9, 149.4, 141.4, 118.8, 76.0, 73.8, 66.3, 60.5,
51.6, 22.4, 14.7, 7.0. Anal. Calcd for C13H17N5O3ꢁ0.3MeOH: C, 53.09; H, 6.10; N,
23.27. Found: C, 53.48; N, 6.28; H, 22.82.
9
7
3
3
Adeno
a
b
c
Effective concentration required for reducing virus level by 50%.
Concentration of compound for 50% cell inhibition without virus.
Selectivity index (SI) = CC50/EC50
.
25. Van Roey, P.; Salerno, J.; Chu, C.; Schinazi, R. Proc. Natl. Acad. Sci. U.S.A. 1989, 86,
3929.
26. Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J. Am. Chem. Soc. 1990,
112, 6127.
A(H1N1), Flu A (H3N2), Flu A (H5N1), VEE, WNV, rhinovirus, mea-
sles, yellow fever, PIV and adeno. The compound was inactive in
most of the viruses with the exceptions of weakly active against vac-
27. MacroModel, V 9.1 Schrodinger Suit 2006; LLC, New York, NY2005.
28. Crystal data of 8: C30H42N10O8, M = 670.74, monoclinic, space group P2(1),
a = 14.573(3), b = 7.951(2), c = 14.888(3) Å, b = 107.93(3)°, V = 1641.2(6) Å3,
T = 293 K, Z = 2, R1 = 0.0329 for 2300 Fo > 4sig(Fo) and 0.0373 for all 2517
data. Crystallographic data has been deposited with Cambridge
Crystallographic Data Centre as supplementary publication number CCDC
816703. These data can be obtained free of charge from www.ccdc.cam.uk/
conts/retrieving.html or from Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge, CB2 1EZ, UK; E-mail: deposit@ccdc.cam.ac.uk.
Programs: HKL Scalepack (Otwinowski & Minor, 1997), Denzo and Scalepak
cinia (EC50 = 42.7
EC50 = 7 M) and adeno (EC50 = 3
in (Table 3).
l
M), measles (NR, EC50 = 9
lM; visual,
l
lM in both NR & visual) as shown
In summary, a novel spiro-carbocyclic adenosine 8 was synthe-
sized and evaluated as a potential antiviral agent. The compound 8
exhibited an interesting anti-HCV activity against both genotype
1A and 1B. The conformation of the novel nucleoside was also
studied by X-ray and calculation. In view of this interesting biolog-
ical activity of the novel nucleoside, the structure–activity relation-
ships as well as further biological studies are warranted.
(Otwinowski
& Minor, 1997), SHELXS-97 (Sheldrick, 1990) and SHELXL-97
(Sheldrick, 1997).
29. Farrugia, L. J. J. Appl. Crystallogr. 1997, 30, 565.
30. Korba, B. E.; Montero, A. B.; Farrar, K.; Gaye, K.; Mukerjee, S.; Ayers, M. S.;
Rossignol, J.-F. Antiviral Res. 2008, 77, 56.
31. Pierra, C.; Amador, A.; Benzaria, S.; Cretton-Scott, E.; D’Amours, M.; Mao, J.;
Mathieu, S.; Moussa, A.; Bridges, E. G.; Standring, D. N.; Sommadossi, J.-P.;
Storer, R.; Gosselin, G. J. Med. Chem. 2006, 49, 6614.
Acknowledgment
This research was supported in part by the US Public Health
Service Grant AI25899 from the National Institute of Allergy and
Infectious Diseases, NIH.