Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides

Article abstract of DOI:10.1080/15257770.2016.1163374

The present study includes the exploration of new possible nucleoside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides (4a–g), which were synthesized by 10–15 synthetic steps and characterized adequately. We report the anti-

Full text of DOI:10.1080/15257770.2016.1163374

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis and Anti-HCV Activity of 4-Methoxy-7H-
Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides
Anandarajan Thiyagarajan, Masaaki Toyama, Masanori Baba, Ashoke Sharon
& Chandralata Bal
To cite this article: Anandarajan Thiyagarajan, Masaaki Toyama, Masanori Baba, Ashoke
Sharon & Chandralata Bal (2016) Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-
d] Pyrimidine Carbocyclic Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 35:6,
305-314, DOI: 10.1080/15257770.2016.1163374
To link to this article: http://dx.doi.org/10.1080/15257770.2016.1163374
Published online: 06 May 2016.
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Date: 23 May 2016, At: 03:20

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
http://dx.doi.org/./..
Synthesis and Anti-HCV Activity of -Methoxy-H-
Pyrrolo[,-d] Pyrimidine Carbocyclic Nucleosides
Anandarajan Thiyagarajana, Masaaki Toyamab, Masanori Babab, Ashoke Sharona,
and Chandralata Bala
aDepartment of Chemistry, Birla Institute of Technology, Mesra, Ranchi, India; bDivision of Antiviral
Chemotherapy, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima,
Japan
ABSTRACT
ARTICLE HISTORY
Received  June 
Accepted  March 
The present study includes the exploration of new possible nucle-
oside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine
carbocyclic nucleosides (4a–g), which were synthesized by 10–15
synthetic steps and characterized adequately. We report the anti-
HCV activities and cytotoxicities of 4a–g. Compound 4a was ana-
lyzed by single crystal X-ray diffraction which showed some puck-
ering in the cyclopentene ring with a 2ꢀ-endo conformation and
anti-base disposition (χ = −125.7°).
KEYWORDS
Carbocyclic nucleosides;
HCV; Selectfluor;
tributylvinyltin;
trimethylsilylacetylene;
H-pyrrolo[,-d]pyrimidine
Introduction
It has been observed that long-term infection due to Hepatitis C virus (HCV)
can lead to chronic liver disease.[1] Standard therapies such as the combination
of peginterferon-alpha (pegIFN-α) with ribavirin/telaprevir/boceprevir have been
found to have significant side-effects.[2] The introduction of US FDA approved
sofosbuvir (Sovaldi) in combination therapy has resulted in successful treatment
without pegIFN-α.[3] However, the high cost of treatment regimen[4] still makes it
desirable to search for new drug candidates. Recently, we reported anti-HCV activ-
ities and cytotoxicities of a new series of 4-methyl-7H-pyrrolo[2,3-d] pyrimidine
carbocyclic nucleosides (I, Figure 1).[5] Though the new compounds were not sig-
nificantly active, most of them were non-toxic up to 100 µM concentration. The
literature survey indicates that a methyl group can form a weak H-bond such as
C–H …O,[6] C–H …π[7] etc. by acting as a hydrogen donor, however, a methoxy
group may act as a hydrogen acceptor (through -O).[8] It is a well-known fact that
minor changes in type and mode of non-covalent interactions significantly affect
ligand–receptor binding and may lead to enhancement in the biological profile of the
ligand. Therefore, in the present study we synthesized 4-methoxy-7H-pyrrolo[2,3-
d]pyrimidine carbocyclic nucleosides (II, Figure 1) in order to explore the
CONTACT Chandralata Bal
cbal@bitmesra.ac.in; Ashoke Sharon
Chemistry, Birla Institute of Technology, Mesra, Ranchi , India.
asharon@bitmesra.ac.in
Department of
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
©  Taylor & Francis Group, LLC

306
A. THIYAGARAJAN ET AL.
Figure . Chemical structure of I (previously reported from our group) and prototypes II for the
present study.
replacement of the methyl group with a methoxy group and compare the anti-HCV
activity of compounds II with their methyl analogs (I).
Results and discussion
Chemistry
The synthesis of 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides
started with the synthesis of carbocyclic sugar (1) in eight steps[5,9] from commer-
cially available d-ribose. 4-Chloro-7H-pyrrolo[2,3-d]pyrimidine (2) was purchased
from CiVenti Chem (India) Private Limited. The 4-chloro group of 2 was replaced
with a methoxy group by refluxing it with KOH in methanol to yield 3a (Scheme 1).
Chloro/bromo/iodo groups were introduced at C-7 of 3a by reacting with respec-
tive N-halosuccinimide to generate 3c–e. The C-7-fluoro base (3b) was synthesized
from the reaction of 2 with selectfluor followed by with NaOMe.
Mitsunobu coupling of 1 with 3a–e yielded protected coupled products, which
were purified and deprotected to yield desired final compounds 4a–e (Scheme 2).
Earlier reported methodology described by our group[5] was followed (Scheme 2)
to synthesize the vinyl (4f) and acetylene (4g) derivatives.
The synthesized compounds were characterized using NMR, mass, UV spectro-
scopic, and melting point analysis. In addition, optically active compounds were
analyzed for specific rotation. The structure of 4a was further confirmed by a sin-
gle crystal X-ray diffraction study.[10] The ORTEP diagram is shown in Figure 2
Scheme . Reagents and conditions: (i) MeOH, KOH, °C,  h; (ii) appropriate N-halosuccinimide,
DMF, rt,  hours; (iii) Selectfluor, CHCN, acetic acid, °C,  hours; (iv) NaOMe, MeOH, °C,  hours.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
307
Scheme . Reagents and conditions: (i) PhP, DIAD, THF, –°C,  hours; (ii) % HCl in CHOH, °C,
 hours; (iii) Pd(PPh), CuI, trimethylsilylacetylene, EtN, DMF, rt,  hours, (iv) KCO, CHOH, rt,
 hours; (v) Pd(PPh), CuI, tributylvinyltin, EtN, DMF, °C,  hours.
with atomic numbering. The crystal structure studies revealed puckering in carbo-
cyclic sugar ring (2ꢀ-endo conformation at C14 atom) with anti-base disposition
(χ = −125.7°; dihedral angle χ: C11–C10–N1–C2).
Anti-HCV activity and cytotoxicity assay
The anti-HCV assay[11,12] of 4a–g was done in the subgenome HCV RNA replicon
cells containing the luciferase gene.[13] The activity and cytotoxicity were evaluated
on the basis of reduction in the luciferase activity and reduction in the number of
viable cells, respectively. From the dose-dependent study results (data not shown),
Figure . ORTEP diagram showing displacement ellipsoid plot (% probability) of X-ray crystal struc-
ture of 4a.

308
A. THIYAGARAJAN ET AL.
Table . Anti-HCV activity of 4a–g and I.
EC (µM)
CC (µM)
ECa (µM) CCa (µM)
4a, R = H
>
>
>
.
.
.
>
>
>
>
>
.
R = H
R = F
>
>
.
>
>
>
>
>
.
4b, R = F
4c, R = Cl
R = Cl
4d, R = Br
4e, R = I
R = Br
.
R = I
.
.
.
4f, R = Vinyl
4g, R = Acetylene
R = Vinyl
>
>
R = Acetylene
>
aThe EC and CC values of I are taken from our previous report for comparative analysis.
EC50 (50% effective concentration) and CC50 (50% cytotoxic concentration) were
calculated. The results are summarized in Table 1.
From the results, it was found that 4d–f have some anti-HCV activity, while 4a–c
and 4g were inactive. Except for 4f, none of the compounds were found to be cyto-
toxic (CC50 values > 100 µM). All the methoxy nucleosides showed similar cytotox-
icity profile as their respective methyl analogs, however, there was no improvement
in anti-HCV activity. In our previous report,[5] we had highlighted that ATP (the
natural nucleoside substrate for HCV polymerase) acquires a 2ꢀ-exo sugar confir-
mation along with anti-base disposition. Therefore, to mimic ATP, the synthesized
nucleosides should also acquire similar confirmation. However, 4a was observed to
acquire 2ꢀ-endo sugar confirmation with anti-base disposition in single crystal X-
ray diffraction analysis (Figure 2) which partially supports the result obtained from
anti-HCV assay.
Experimental
General methods
All reactions were carried out in oven-dried glasswares (120°C) under an atmo-
sphere of nitrogen unless indicated otherwise. Most of the chemicals were pur-
chased from Merck or Sigma–Aldrich or Spectrochem Chemical Companies as
per availability. d-ribose was purchased from Carbosynth Limited. 4-Chloro-7H-
pyrrolo[2,3-d]pyrimidine was purchased from CiVenti Chem (India) Private Lim-
ited. Melting points were recorded on Veego melting point apparatus. Analytical
thin layer chromatography (TLC) was performed on precoated plates (silica gel 60
F-254) purchased from Merck Inc. Purification by gravity column chromatography
was carried out with silica gel (100–200 mesh). UV–visible spectra were recorded
on Perkin-Elmer System. The optical rotations of optically active compounds were
1
measured on Rudolph Autopol I. H (400 MHz) and 13C (100 MHz) NMR were

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
309
obtained from a Varian or Bruker spectrometer using CDCl3 or DMSO-d6 as sol-
vents. Peaks are recorded with the following abbreviations: s, singlet; bs, broad sin-
glet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling constant (hertz).
Synthesis of 3a
To a solution of 2 (9.82 mmol) in dry methanol at room temperature (rt), KOH
(19.65 mmol) was added under nitrogen and refluxed at 80°C for 4 hours. After
completion of reaction (monitored by TLC), the reaction mixture (RM) was cooled,
poured into ice water and extracted with ethyl acetate. The organic layer was
washed with brine, dried with anhydrous Na2SO4, and concentrated. The crude
was purified by column chromatography on silica gel (eluting up to 5% methanol
in dichloromethane) to yield 3a as white crystalline solid.
-Methoxy-H-pyrrolo[,-d]pyrimidine (a)
1
Yield: 85%; mp: 209–211°C; MS-ESI (m/z): [M++1] 149.9; H NMR (400 MHz,
DMSO-d6): δ 4.03 (s, 3H, CH3), 6.46–6.48 (d, J = 3.6 Hz, 1H, ArH), 7.34–7.36 (d,
J = 3.6 Hz, 1H, ArH), 8.37 (s, 1H, ArH), 12.04 (bs, 1H, NH); UV (MeOH) λ
:
max
263.1 nm.
Synthesis of 3c–e
To a solution of 3a (1.67 mmol) in dry DMF, appropriate N-halosuccinimide
(1.81 mmol) was added and stirred under nitrogen at room temperature for 4 hours.
The RM was poured into ice water and stirred for 30 minutes. The precipitate was
filtered, washed with water, and purified by column chromatography on silica gel
(eluting with up to 5% methanol in dichloromethane) to get 3c–e.
-Chloro--methoxy-H-pyrrolo[,-d]pyrimidine (c)
Yield: 78%; mp: 249–251°C; MS-ESI (m/z): [M+] 183.8, [M++2] 185.8; 1H NMR
(400 MHz, DMSO-d6) δ 4.05 (s, 3H, CH3), 7.53 (s, 1H, ArH), 8.41 (s, 1H, ArH),
12.33 (bs, 1H, NH).
-Bromo--methoxy-H-pyrrolo[,-d]pyrimidine (d)
Yield: 80%; mp: 235–237°C; MS-ESI (m/z): [M+] 227.7; [M++2] 229.8; 1H NMR
(400 MHz, DMSO-d6): δ 4.05 (s, 3H, CH3), 7.56 (s, 1H, ArH), 8.39 (s, 1H, ArH),
12.40 (bs, 1H, NH).
-Iodo--methoxy-H-pyrrolo[,-d]pyrimidine (e)
1
Yield: 83%; mp: 218–220°C; MS-ESI (m/z): [M++1] 275.7; H NMR (400 MHz,
DMSO-d6): δ 4.04 (s, 3H, CH3), 7.56 (s, 1H, ArH), 8.38 (s, 1H, ArH), 12.38 (bs, 1H,
NH).

310
A. THIYAGARAJAN ET AL.
Synthesis of 3b
A reaction mixture consisting of 2 (3.32 mmol) and Selectfluor (4.97 mmol) in dry
acetonitrile:acetic acid (5:1) was heated at 70°C under nitrogen for 6 h. The RM
was cooled to room temperature and solvent was removed under vacuum. Crude
was extracted with DCM, organic layer dried with Na2SO4 and concentrated. The
residue obtained was purified by column chromatography on silica gel (eluting up
to 30% Ethyl acetate in hexane) to get an off-white solid which was dissolved in
dry methanol and NaOMe (2.55 mmol, calculated with respect to 2) was added
under nitrogen. The resulting RM was heated at 80°C for 2 hours and then cooled
to room temperature, poured into ice water and extracted with ethyl acetate. The
organic layer was washed with brine, dried with anhydrous Na2SO4 and concen-
trated. The crude was purified by column chromatography on silica gel (eluting up
to 10% methanol in dichloromethane).
-Fluoro--methoxy-H-pyrrolo[,-d]pyrimidine (b)
1
Yield: 65%; mp: 213–215°C; MS-ESI (m/z): [M+-1] 166.0; H NMR (400 MHz,
DMSO-d6): δ 4.05 (s, 3H, CH3), 7.32 (s, 1H, ArH), 8.37 (s, 1H, ArH), 11.90 (bs, 1H,
NH); UV (MeOH) λ max: 262.2 nm.
Synthesis of 4a–e
DIAD (3.75 mmol) was added dropwise to a stirring mixture of appropriate 3a–e
(1.5 mmol), 1 (1.57 mmol) and Ph3P (3.75 mmol) in THF at 0°C under nitro-
gen and stirring continued for 2 hours. Solvent was evaporated under reduced
pressure and crude was purified by column chromatography on silica gel by elut-
ing up to 30% ethyl acetate in hexane to get coupled products. The deprotec-
tion of coupled products was carried out by heating at 60°C in 10% methano-
lic HCl for 5 hours. After reaction completion, the RM was neutralized by
NaHCO3 and purified by silica gel chromatography (10% methanol in DCM) to get
4a–e.
(S,R,R)--(hydroxymethyl)--(-methoxy-H-pyrrolo[,-d]pyrimidin--
yl)cyclopent--ene-,-diol (a)
Yield: 82%; mp: 164–166°C; MS-ESI (m/z): [M++1] 277.9; [α]D20: −114.8 (c
0.12 MeOH); UV (MeOH) λ max 261.1 nm; 1H NMR (400 MHz, DMSO-d6): δ 4.04
(s, 3H, CH3), 4.13–4.17 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.44–4.42 (t, J = 5.6 Hz, 1H, 3ꢀ-
CH), 4.89–4.92 (m, 1H, OH), 4.96–4.94 (d, J = 6.4 Hz, 1H,OH), 5.04–5.02 (d, J =
6.8 Hz, 1H, OH), 5.62–5.65 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 6.55–6.54 (d, J = 3.6 Hz, 1H,
ArH), 7.31–7.30 (d, J = 3.6 Hz, 1H, ArH), 8.42 (s, 1H, ArH): 13C NMR (100 MHz,
DMSO-d6) δ 53.81, 59.00, 64.71, 72.76, 77.67, 98.40, 105.28, 124.51, 125.06, 150.53,
150.71, 152.09, 162.67.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
311
(S,R,R)--(-fluoro--methoxy-H-pyrrolo[,-d]pyrimidin--yl)--
(hydroxymethyl) cyclopent--ene-,-diol (b)
23
Yield: 75%; mp: 168–170°C; MS-ESI (m/z): [M+] 296.2; [α]D −120.9 (c 0.16
MeOH); UV (MeOH) λ max 259.8 nm, 283.2 nm; 1H NMR (400 MHz, DMSO-d6):
δ 4.05 (s, 3H, CH3), 4.09–4.12 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.41–4.38 (t, J = 5.6 Hz, 1H,
3ꢀ-CH), 4.89–4.91 (m, 1H, OH), 4.95–4.96 (d, J = 6.4 Hz, 1H,OH), 5.04–5.06 (d, J
= 6.8 Hz, 1H,OH), 5.58–5.67 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 7.30 (s, 1H, ArH), 8.42 (s, 1H,
ArH).
(S,R,R)--(-chloro--methoxy-H-pyrrolo[,-d]pyrimidin--yl)--
(hydroxymethyl) cyclopent--ene-,-diol (c)
21
Yield: 73%; mp: 189–191°C; MS-ESI (m/z): [M+] 311.8; [M++2] 313.8; [α]D
−131.6 (c 0.13 MeOH); UV (MeOH) λ max 259.8 nm, 283.2 nm1H NMR (400 MHz,
DMSO-d6): δ 4.05 (s, 3H, CH3), 4.12–4.16 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.41–4.43 (t, J
= 5.6 Hz, 1H, 3ꢀ-CH), 4.86–4.88 (m, 1H, OH), 4.93–4.95 (d, J = 6.0 Hz, 1H,OH),
5.04–5.06 (d, J = 7.2 Hz, 1H, OH), 5.61–5.63 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 7.47 (s, 1H,
ArH), 8.44 (s, 1H, ArH): 13C NMR (100 MHz, DMSO-d6) δ 54.20, 59.02, 64.99,
72.72, 77.46, 102.10, 102.96, 122.43, 123.99, 150.69, 151.10, 151.65, 162.61.
(S,R,R)--(-bromo--methoxy-H-pyrrolo[,-d]pyrimidin--yl)--
(hydroxymethyl) cyclopent--ene-,-diol (d)
21
Yield: 65%; mp: 208–210°C; MS-ESI (m/z): [M+] 355.8, [M++2] 357.7; [α]D
−124.4 (c 0.16 MeOH); UV (MeOH) λ max260.2 nm, 284.8 nm; 1H NMR (400 MHz,
DMSO-d6) δ 4.05 (s, 3H, CH3), 4.11–4.17 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.41–4.44 (t,
J = 5.6 Hz, 1H, 3ꢀ-CH), 4.86–4.88 (m, 1H, OH), 4.93–4.95 (d, J = 6.0 Hz, 1H, OH),
5.04–5.05 (d, J = 6.8 Hz, 1H, OH), 5.61–5.63 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 7.50 (s, 1H,
ArH), 8.44 (s, 1H, ArH).
(S,R,R)--(hydroxymethyl)--(-iodo--methoxy-H-pyrrolo[,-d]pyrimidin--yl)
cyclopent--ene-,-diol (e)
21
Yield: 72%; mp: 218–220°C; MS-ESI (m/z): [M++1] 403.7; [α]D −126.2 (c 0.16
MeOH); UV (MeOH) λ
259.5 nm, 285 nm; 1H NMR (400 MHz, DMSO-d6) δ
max
4.05 (s, 3H, CH3), 4.11–4.17 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.41–4.44 (t, J = 5.6 Hz, 1H,
3ꢀ-CH), 4.86–4.89 (m, 1H, OH), 4.93–4.95 (d, J = 6.0 Hz, 1H,OH), 5.03–5.05 (d, J
= 6.4 Hz, 1H,OH), 5.61–5.67 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 7.49 (s, 1H, ArH), 8.42 (s, 1H,
ArH).
Synthesis of 4f
A suspension of 4e (0.25 mmol), Pd(PPh3)4 (0.02 mmol), CuI (0.05 mmol), and
Et3N (0.77 mmol) in anhydrous DMF was purged with nitrogen at room tempera-
ture. Tributylvinyltin (2.58 mmol) was added under nitrogen and the mixture was

312
A. THIYAGARAJAN ET AL.
stirred at 50°C for 6 hours. The completion of reaction was checked by TLC, sol-
vent was removed and residue was purified on silica gel (100–200 mesh) column
chromatography by eluting with 10% methanol in DCM.
(S,R,R)--(hydroxymethyl)--(-methoxy--vinyl-H-pyrrolo[,-d]pyrimidin--yl)
cyclopent--ene-,-diol (f)
21
Yield 75%; mp: 145–147°C; MS-ESI (m/z): [M++1] 303.9; [α]D −164.3 (c 0.11
MeOH); UV (MeOH) λ max 277 nm: 1H NMR (400 MHz, DMSO-d6) δ 4.06 (s, 3H,
CH3), 4.13–4.19 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.43–4.46 (m, 1H, 3ꢀ-CH), 4.88–4.91 (m,
1H, OH), 4.94–4.96 (d, J = 6.0 Hz, 1H, OH), 5.04–5.06 (d, J = 6.8 Hz, 1H, OH),
5.11–5.15 (m, 1H, vinyl-CH2), 5.63–5.66 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 5.79–5.84 (m, 1H,
vinyl-CH2), 6.86–6.93 (m, 1H, vinyl-CH), 7.56 (s, 1H, ArH), 8.41 (s, 1H, ArH).
Synthesis of 4g
A suspension of 4e (0.25 mmol), Pd(PPh3)4 (0.02 mmol), CuI (0.05 mmol), and
Et3N (0.77 mmol) in anhydrous DMF was purged with nitrogen at room temper-
ature for 10 hours. Trimethylsilylacetylene (2.58 mmol) was added under nitrogen
and the mixture was stirred at room temperature. The completion was checked by
TLC, solvent was removed and residue was purified on silica gel (100–200 mesh)
chromatography by eluting with 10% methanol in DCM to give trimethylsilyl pro-
tected compound in 80% yield. The deprotection was done by stirring in methanol
and K2CO3 (0.1 mmol) at room temprature for 4 hours and purified by silica gel
chromatography by eluting up to 10% methanol in DCM to get 4g as a light brown
solid in 75% yield.
(S,R,R)--(-ethynyl--methoxy-H-pyrrolo[,-d]pyrimidin--yl)--
(hydroxymethyl) cyclopent--ene-,-diol (g)
21
Yield: 71%; mp: 131–133°C; MS-ESI (m/z): [M++1] 301.9; [α]D −157.2 (c 0.17
MeOH); UV (MeOH) λmax 268 nm; 1H NMR (400 MHz, DMSO-d6) δ 4.05 (s, 1H,
Acetylene-CH), 4.06 (s, 3H, CH3), 4.12–4.19 (m, 3H, 5ꢀ-CH2, 2ꢀ-CH), 4.43–4.46 (m,
1H, 3ꢀ-CH), 4.86–4.89 (m, 1H, OH), 4.94–4.96 (d, J = 6.0 Hz, 1H, OH), 5.05–5.07
(d, J = 7.2 Hz, 1H,OH), 5.61–5.63 (m, 2H, 1ꢀ-CH, 6ꢀ-CH), 7.67 (s, 1H, ArH), 8.45
(s, 1H, ArH).
Anti-HCV assay
The anti-HCV assay of 4a–g was carried out in subgenome HCV RNA replicon cells
containing the luciferase gene. All compounds were dissolved in dimethyl sulfoxide
(DMSO) at a concentration of 20 mM or higher to exclude the cytotoxicity of DMSO
and stored at −20°C until use. Huh-7 cells containing self-replicating subgenomic
replicons with a luciferase reporter, LucNeo#2,[13] were grown and cultured in Dul-
becco’s modified Eagle medium with high glucose (Gibco/BRL) supplemented with
10% heat-inactivated fetal bovine serum (Gibco/BRL), 100 U/mL penicillin G, and

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
313
100 µg/mL streptomycin. LucNeo#2, were maintained in culture medium contain-
ing 1 mg/mL G418 (Nakarai Tesque). The anti-HCV activity of the test compounds
was determined in LucNeo#2 cells by the previously described method with some
modifications.[11] Briefly, the cells (5 × 103 cells/well) were cultured in a 96-well
plate in the absence of G418 and in the presence of various concentrations of the
compounds. After incubation at 37°C for 3 days, the culture medium was removed,
and the cells were washed twice with phosphate-buffered saline (PBS). Lysis buffer
was added to each well, and the lysate was transferred to the corresponding well of
a non-transparent 96-well plate. The luciferase activity was measured by addition of
the luciferase reagent in a luciferase assay system kit (Promega) using a luminome-
ter with automatic injectors (Berthold Technologies). The number of viable cells was
determined by a dye method using the water-soluble tetrazolium Tetracolor One
(Seikagaku Corporation), according to the manufacturer’s instructions.
®
Conclusion
The present study includes synthesis of new possible nucleoside mimetic based on 4-
methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleoside (4a-g)involving 10–
15 synthetic steps with adequate characterization including single crystal analysis of
one of them. Further, the anti-HCV activities and cytotoxicities of 4a–g were evalu-
ated. Although the new analogs (4a–g) did not show notable improvement in their
anti-HCV activity profile in comparison to the previously explored methyl deriva-
tive, the maintenance of significant cytotoxicity profile opens a scope for further
exploration through modification in base and carbocyclic sugar.
Acknowledgments
This research received funding from UGC, New Delhi, India (F. No. 39-705/2010). The authors
acknowledge Dr. Reddy’s Institute of Life Sciences, Hyderabad for NMR/Mass Facility, Central
Instrument Facility, BIT Mesra for analytical support and Agilent Technologies for X-ray data
collection and structure solution. A.S. and C.B. acknowledge DST FIST support.
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