A conformational mimetic approach for the synthesis of carbocyclic nucleosides as anti-HCV leads

Article abstract of DOI:10.1002/cmdc.201300277

Computer-aided approaches coupled with medicinal chemistry were used to explore novel carbocyclic nucleosides as potential anti-hepatitisC virus (HCV) agents. Conformational analyses were carried out on 6-amino-1H-pyrazolo[3,4-d]pyrimidine (6-APP)-based carbocyclic nucleoside analogues, which were considered as nucleoside mimetics to act as HCV RNA-dependent RNA polymerase (RdRp) inhibitors. Structural insight gained from the modeling studies revealed the molecular basis behind these nucleoside mimetics. The rationally chosen 6-APP analogues were prepared and evaluated for anti-HCV activity. RdRp SiteMap analysis revealed the presence of a hydrophobic cavity near C7 of the nucleosides; introduction of bulkier substituents at this position enhanced their activity. Herein we report the identification of an iodinated compound with an EC₅₀ value of 6.6μM as a preliminary anti-HCV lead.

Full text of DOI:10.1002/cmdc.201300277

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DOI: 10.1002/cmdc.201300277
A Conformational Mimetic Approach for the Synthesis of
Carbocyclic Nucleosides as Anti-HCV Leads
[
a]
[a]
[b]
[a]
Mohan Kasula, Tuniki Balaraju, Massaki Toyama, Anandarajan Thiyagarajan,
[a]
[b]
[a]
Chandralata Bal, Masanori Baba, and Ashoke Sharon*
Computer-aided approaches coupled with medicinal chemistry
were used to explore novel carbocyclic nucleosides as poten-
tial anti-hepatitis C virus (HCV) agents. Conformational analyses
were carried out on 6-amino-1H-pyrazolo[3,4-d]pyrimidine (6-
APP)-based carbocyclic nucleoside analogues, which were con-
sidered as nucleoside mimetics to act as HCV RNA-dependent
RNA polymerase (RdRp) inhibitors. Structural insight gained
from the modeling studies revealed the molecular basis
behind these nucleoside mimetics. The rationally chosen 6-APP
analogues were prepared and evaluated for anti-HCV activity.
RdRp SiteMap analysis revealed the presence of a hydrophobic
cavity near C7 of the nucleosides; introduction of bulkier sub-
stituents at this position enhanced their activity. Herein we
report the identification of an iodinated compound with an
EC50 value of 6.6 mm as a preliminary anti-HCV lead.
Introduction
Hepatitis C virus (HCV) is considered major health threat, and
chronic infection can lead to liver diseases such as cirrhosis
[
1]
and hepatocellular carcinoma. Among the number of direct-
acting antiviral compounds in clinical trials or approved for use
in the treatment of chronic HCV, members of the nucleoside
drug class have pan-genotypic activity. The degradation of nat-
ural nucleosides to ribose may be prevented by replacing the
[2]
oxygen atom of the sugar ring with a methylene group.
Because of these properties, carbocyclic nucleosides have re-
[3]
ceived much attention as potential chemotherapeutic agents.
Furthermore, the discovery of abacavir and entecavir (Figure 1)
for the treatment of HIV and HBV, respectively, prompted us to
explore newer carbocyclic nucleosides for the treatment of
HCV. Various structural modifications have been reported in
Figure 1. Structures of abacavir (approved anti-HIV drug), entecavir (ap-
proved anti-HBV drug), neplanocin A (NPA), 7-deaza-NPA, and target carbo-
cyclic nucleosides based on 6-amino-1H-pyrazolo[3,4-d]pyrimidine (6-APP, I).
[4]
the discovery of new molecules with improved efficacy. Ne-
planocin A (NPA, Figure 1), a carbocyclic nucleoside analogue
of adenine with a cyclopentene ring in place of ribose, has
broad-spectrum antiviral activity. However, its cytotoxicity pre-
[
5]
cluded its development as a drug. Despite several modifica-
tions in the cyclopentene ring of NPA, none of the resulting
“anti” conformation of a base with a hydrogen bond donor at
the 6-position and a 2’-exo conformation for the sugar are con-
[
6]
analogues showed promising activity. However, base modifi-
cation (7-deaza-adenine) by Chu and co-workers resulted in
[8]
ducive to anti-HCV activity. 1H-Pyrazolo[3,4-d]pyrimidine (PP)
nucleosides shown to acquire the “anti” glycosyl conforma-
[
7]
significant anti-HCV activity, which opened the door to ex-
plore other bioisosteres of adenine. In continuation of our nu-
cleoside mimetic design process, we recently reported that the
[9]
tion have been explored extensively for a range of antiviral
[10]
activities.
was extended to select 6-amino-1H-pyrazolo[3,4-d]pyrimidine
6-APP) as a base coupled with a cyclopentene ring in order to
Therefore, the conformational mimetic approach
(
[
a] M. Kasula, T. Balaraju, A. Thiyagarajan, Dr. C. Bal, Dr. A. Sharon
Department of Applied Chemistry, Birla Institute of Technology
Mesra, Ranchi, Jharkhand, 835215 (India)
explore a new class of carbocyclic nucleoside analogues (I,
Figure 1) as potential anti-HCV agents.
E-mail: asharon@bitmesra.ac.in
[b] Dr. M. Toyama, Dr. M. Baba
Division of Antiviral Chemotherapy, Center for Chronic Viral Disease
Kagoshima University, Sakuragaoka, Kagoshima (Japan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300277.
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Results and Discussion
Conformational studies
We recently reported the use of a 3D model of HCV RNA-de-
pendent RNA polymerase (HCV RdRp) complex in our nucleo-
side inhibitor design process and described certain conforma-
[
8]
tional requirements for anti-HCV activity. Previously, Marquez
et al. also suggested that specific nucleoside conformations are
necessary for binding with viral polymerases in order to block
[
11]
their function. Therefore, a well-defined shape analysis is re-
quired for correlating conformation with biological activity.
The 6-APP carbocyclic nucleosides are structurally similar to
NPA and 7-deaza-NPA (Figure 1). Recently, Chu and colleagues
reported that 7-deaza-NPA has significant activity against HCV
[
7]
Figure 3. The top five conformational superimposition poses of 7-bromo-
substituted 6-APP carbocyclic nucleoside.
(2.5 mm). Therefore, we analyzed the conformational behav-
iors of these three different compound classes (Figure 2).
Among the top-five conformations of NPA (Figure 2a), four
were found to be in the syn base disposition, whereas one
showed an anti base disposition; all five conformers were ob-
served to have a roughly planar sugar pucker. Both 7-deaza-
NPA (Figure 2b) and the unsubstituted 6-APP carbocyclic nu-
cleoside (Figure 2c) had four anti and one syn disposition. In
addition, all the anti conformers of 7-deaza-NPA and the ana-
lyzed 6-APP analogue were observed in the 2’-exo sugar con-
formation. Overall, these results highlight the conformational
similarity between 7-deaza-NPA and 6-APP carbocyclic nucleo-
sides.
inhibitors may show better HCV RdRp inhibition if they have
the tendency to adopt an anti base disposition along with
[8]
a 2’-exo sugar conformation. Therefore, we considered 6-APP
carbocyclic nucleoside analogues as suitable candidates for de-
velopment into a new class of anti-HCV compounds.
Molecular docking studies
These molecules were also assessed for their ability to bind
[12]
HCV RdRp. A previously reported model was further refined
and optimized by using a recently reported X-ray crystal struc-
We also studied the conformational properties of prototype
I analogues prior to synthesis. The conformational superposi-
tion of the 7-bromo-6-APP analogue is shown in Figure 3. In-
terestingly, it shows 100% anti base disposition with a 2’-exo
sugar conformation. The C7-chloro and -iodo compounds had
shown the same conformational behavior as the C7-bromo an-
alogue. However, the C7-fluoro compound proved to be simi-
lar to the unsubstituted analogue. Cluster analysis of NPA (Fig-
ure 2a) suggested an 80% syn disposition with planar sugar
puckering. These results clearly demonstrate the distinct con-
formational behavior of pyrazolo[3,4-d]pyrimidine analogues I
relative to NPA. In our earlier studies, we found that nucleoside
[13]
ture of an HCV primer–template complex.
The template,
primer, and metal ions were extracted from the crystal struc-
ture of the HCV RdRp complex during construction of the 3D
model. The natural ATP substrate was docked into the nucleo-
side active site to generate the HCV RdRp–ATP model. The
ATP-docked model was further subjected to 5 ns molecular dy-
namics (MD) simulation. The final HCV RdRp model, consisting
of the template–primer complex (2:3), ATP, and two magnesi-
um ions, was selected. The nucleoside analogues must be con-
verted into their triphosphate form by kinase activity to act as
competitive inhibitors of the natural ATP substrate. For effec-
Figure 2. Top five conformers of a) NPA, b) 7-deaza-NPA, and c) 6-APP carbocyclic nucleoside (I, R=H) for conformational comparison.
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tive comparison with ATP, the triphosphate analogues of com-
pounds 6a–d were therefore built and docked into the active
site of HCV RdRp. The superimposed docking poses of 6a–d
triphosphates with ATP (Figure 4) suggests their ability to
mimic ATP to act as HCV RdRp inhibitors.
clearly indicate a difference, suggesting that the 6-APP core is
more suitable as an HCV RdRp inhibitor. Moreover, substitution
at C7 with bulkier groups (Cl, Br, I) provided a better energetic
profile than that of the unsubstituted analogue 6a. It also indi-
cates the possibility of a hydrophobic cavity in the HCV RdRp
active site. SiteMap analysis (Figure 5), carried out in continua-
tion, revealed the hydrophobic nature (yellow) around the 6-
APP base; however, other sub-sites were found to be charged
in nature (blue, red, or pink mesh). This analysis showed
a cavity available proximal to the C7 position of 6-APP in the
hydrophobic region (Figure 5, yellow mesh).
[
14]
The Glide module of Schrçdinger Suite was used for mo-
lecular docking to study the binding mode of compounds 6a–
d and to evaluate their docking scores. The Glide docking
[
15]
scores, conformational, and drug-like properties are summar-
ized in Table 1. Chemically and structurally, the 6-APP core,
which is a single-atom-replacement bioisosteric analogue of
NPA, appears to be very similar to NPA. However, the docking
score (G Score) and free energy of binding (DGbind) (Table 1)
The 6-APP analogues 6a–d were found mostly in the anti
conformation (Table 1, c=À111), which supports the observa-
tions from conformational stud-
ies (Figure 3). Furthermore, the
degree of sugar puckering was
observed to be higher (u=22.2)
than in NPA (u=15.6). Overall,
a
higher level of puckering
along with a 2’-exo conformation
and anti base disposition can be
considered as features of the
best conformer to fit into the
HCV RdRp active site. The Qik-
Prop module of Schrçdinger was
used for analyzing properties as
they relate to Lipinski’s rule of
[16]
five as well as Jorgensen’s rule
[17]
of three,
and none of the
compounds violated either set
of guidelines (Table 1). Further-
more, these molecules were
found to be acceptable in terms
of aqueous solubility and oral
absorption criteria (Table 1).
Thus, the preliminary computa-
tional studies provided a strong
rationale to consider this new
class of 6-APP-based carbocyclic
nucleosides for synthesis and
anti-HCV activity evaluations.
Chemistry and anti-HCV
activity
The key cyclopentene intermedi-
ate 1, was prepared as a single
isomer as per a method reported
[18]
earlier
in eight consecutive
steps by starting from commer-
[8]
cially available d-ribose. 6-APP
2) was synthesized by starting
(
[19]
Figure 4. a) Superimposed docked poses of the triphosphate forms of 6a–d and ATP in the active site of HCV-
RdRp. The image shows interactions of two magnesium ions with major residues (Asp220, Asp318, and Asp319)
and with the phosphates (Pa, Pb, and Pg) of ATP or 6a–d. b) Active site binding interactions of 6d. Metal ions
from malononitrile, and N-tert-
butoxycarbonyl (Boc) was used
for protecting the amino group.
For this purpose a solution of 2
and di-tert-butyl dicarbonate
(MGN) coordinate with phosphate group oxygen atoms of 6d triphosphate; hydrogen bonds are shown as
dotted arrows, and the p–cation interaction between Arg158 and the pyrazole ring is shown by the thick line
flanked by filled circles.
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Table 1. Molecular docking scores, pseudorotation analysis from conformational search, and drug-like properties of 6a–d in comparison with NPA.
[
a]
[b]
Compd
Pseudorotation Analysis
Sugar Puckering
Binding Score
Drug Like Properties
Human Oral Absorption [%] QPlogS
[
b]
[c]
[d]
Base Disposition [%] G Score
DGbind
Rule Violations
[
e]
[f]
#
5
#3
NPA
g: À57, c: À70.2,u: 15.6, P: 344.6
g: À57, c: À111, u: 22.2, P: 343
g: À57, c: À111, u: 22.2, P: 343
g: À57, c: À111, u: 22.2, P: 343
g: À57, c: À111, u: 22.2, P: 343
syn (80%)
anti (80%)
anti (100%)
anti (100%)
anti (100%)
À12.5
À12.8
À13.1
À13.2
À13.2
À64.7
À76.2
À86.9
À87.9
À88.4
52.0
53.6
58.1
58.8
59.4
À1.86
À1.97
À2.48
À2.55
À2.64
0
0
0
0
0
0
0
0
0
0
6
6
6
6
a
b
c
d
[a] g=dihedral angle to show the 5’-OH orientation, c=dihedral angle to describe the base disposition (syn or anti) relative to the central five-membered
ring, u=planarity of central five-membered ring, P=pseudorotation phase angle to describe the overall conformation (P=08 corresponds to north confor-
mation, whereas P=1808 corresponds to south conformation). [b] Glide Score: the minimized poses are rescored using Schrçdinger’s proprietary scoring
function, and the binding affinity can be estimated by G Score. [c] MM-GBSA Score: DGbind =Ecomplex(minimized)À[Eligand(from minimized complex) +Ereceptor]; DGbind is the
ligand–receptor interaction energy of the complex. [d] From À6.5 to 0.5. [e] Number of violations of Lipinski’s rule of five: M
r
<500, QPlogPoctanol/water <5,
À1
HBDꢀ5, HBAꢀ10. [f] Number of violations of Jorgensen’s rule of three: QPlogS>5.7, QPPCaco>22 nms , primary metabolites <7. Compounds with
fewer violations of both sets of rules are more likely to be orally available.
7
of the N8-glycosylated product
showed NOEs with protons of
[22]
the sugar ring. Therefore, we
performed conformational
a
search analysis of N8 and N9 iso-
mers that could be formed
during the coupling of 3a with
1
to calculate the possible dis-
tances (Figure 6) between H-7
and cyclopentene ring protons
(
olefinic and protons above the
plane i.e., H2’ and H3’). We
found that the distances are in
the NOE range for the N8
isomer, whereas they are out of
NOE range for the N9 isomer
(
Figure 6). Finally, scrutinizing
the spatial distance of the pro-
tecting groups (trityl and Boc)
suggests that the N8 isomer is
sterically unfavored, as the two
groups may undergo steric
clash. Therefore, we assigned
Figure 5. Various regions of the nucleoside active sites are shown in different mesh colors. Hydrophobic regions:
yellow, hydrogen bond donors: blue, hydrogen bond acceptors: red, metal binding: pink.
[
(Boc) O] in THF was stirred at room temperature in the pres-
the single products 5a–d as N9 glycons. This regioselectivity
may be attributed to the steric hindrance experienced in for-
mation of the N8 isomer. The structural characterizations were
also confirmed by NMR and COSY analysis. The 2’-H and 3’-H
are in cis; accordingly, the NMR coupling constant was found
to be 5.5 Hz.
2
ence of DMAP as catalyst (Scheme 1). Upon completion of the
reaction, excess THF was removed, and the crude was dis-
solved in methanol and treated with saturated aqueous
sodium bicarbonate at room temperature for 3 h to obtain the
di-Boc derivative 3a.
The reaction of 2 with respective N-halosuccinimides in DMF
generated 7-halogenated derivatives 4a–c. A similar procedure
as described above was followed to furnish the di-Boc deriva-
tives 3b–d. Mitsunobu coupling of 3a–d with 1 yielded 5a–d
as single products (Scheme 2). The UV spectra for compounds
5
a–d were very similar to those of their respective Boc-pro-
[
20]
tected aglycons 3a–d, indicating N9 glycosylation.
In the case of N8 glycosylation, a bathochromic shift should
[
21]
be observed. Furthermore, H-7 of 5a was irradiated in a NOE
experiment, and no correlation with any sugar ring protons
was observed. Seela et al. reported earlier that irradiation of H-
Scheme 1. Synthesis of C7-substituted and Boc-protected 6-APP bases. Re-
agents and conditions: a) (Boc)
2 3
O, DMAP, THF, RT, 6 h; b) sat. NaHCO , MeOH,
RT, 3 h; c) NCS/NBS/NIS, DMF, RT, 4 h.
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replicon cells containing the luci-
[23]
ferase gene LucNeo#2.
The
anti-HCV activities of test com-
pounds were determined from
the decrease in luciferase activi-
ty. Cytotoxicity was evaluated
from the decrease in viable cell
numbers by using a tetrazolium
dye method. EC50 and CC50
values were calculated, and the
results are shown in Figure 7
and summarized in Table 2. The
phenanthridinone derivative KZ-
Scheme 2. Synthesis of pyrazolo[3,4-d]pyrimidine-based carbocyclic nucleoside analogues 6a–d. Reagents and
conditions: a) Ph P, DIAD, THF, 0–58C, 2 h; b) 10% conc. HCl in CH OH, 608C, 5 h.
3 3
16 was used as a reference com-
The deprotection of 5a–d was carried out by holding them
at reflux in 10% concentrated hydrochloric acid in methanol to
yield the targeted carbocyclic nucleosides 6a–d. Anti-HCV
assays of 6a–d were carried out in the sub-genome HCV RNA
pound, which was recently reported to have selective anti-HCV
[23]
activity.
From the results, compounds 6a and 6b were found to be
inactive. It was interesting to find that the introduction of bulk-
ier halogen atoms at C7 markedly increased the ac-
tivity with respect to size, along with better selectivi-
ty.
Conclusions
A conformational mimetic approach was implement-
ed to select 6-APP carbocyclic nucleoside analogues
for synthesis. The conformational search, active site
analysis, and molecular docking studies provided the
rationale to proceed with synthesis. The novel 6-APP-
based carbocyclic nucleosides 6a–d were successfully
prepared, followed by anti-HCV evaluation on repli-
con cells. It was found that the introduction of bulky
groups at C7 enhances activity. Compound 6d, iodi-
nated at C7, showed moderate activity (EC : 6.6 mm)
50
with a selectivity index of 6.6, and was therefore con-
sidered a preliminary lead. However, the lack of po-
tential activity in these compounds may be due to
poor phosphorylation in replicon cells or the inability
of the triphosphate forms to be recognized by RdRp.
Therefore, additional experimental studies are neces-
sary to gain knowledge into their mode of action.
The results of this work set the groundwork for more
detailed studies aimed at optimizing this compound
class to achieve better selectivity as potential direct-
acting antiviral agents.
Experimental Section
Molecular modeling
ATP was considered as a reference molecule for compari-
son against synthesized analogues. The triphosphate an-
alogues of 6a–d can be considered as nucleoside tri-
phosphate mimics (NTMs) and were prepared with the
builder module of Maestro followed by energy minimiza-
tion (EM) through the Macromodel module. EM calcula-
tions were performed with the MMFFs force field for
Figure 6. a) Conformational studies carried out to demonstrate intramolecular atomic dis-
tances (~3.6 ꢁ), which illustrate the possibility for NOE in the N8 isomer only. b) The
large interatomic distances (>4.6 ꢁ) between C7ÀH (base) and the 2’, 3’, and 6’ CH
groups of the cyclopentene ring may not give rise to NOE for the N9 isomer.
5
000 steps or until the energy difference between subse-
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ed for molecular docking studies.
The nature of active site (hydro-
phobic, hydrophilic, metal binding,
etc.) was analyzed by SiteMap
analysis, using the default method
of the SiteMap module of Schrç-
dinger. A receptor grid was gener-
ated around ATP (the natural nu-
cleoside triphosphate) by using
the Glide receptor grid generation
utility. Molecular docking studies
were conducted with NTMs as
ligand at the predefined receptor
grid using the extra precision (XP)
mode of Glide to investigate the
top-five poses and binding score.
The top-docked poses were evalu-
ated using Prime with MMGBSA
free energy of binding (DGbind) to
estimate the NTM–HCV RdRp inter-
action energy (Table 1). The
MMGBSA scoring calculates the
difference between the energy of
the NTM-bound complex and the
sum of the energies of unbound
HCV RdRp: Ebinding =[EcomplexÀ(EHCV–
Figure 7. Anti-HCV activity of 6a–d in replicon cells. Cell viabilities (light grey) and luciferase activities (dark grey)
are shown. All experiments were carried out in duplicate, and data shown are from single representative experi-
ments.
+
ENTM)].
RdRp
Table 2. Anti-HCV activity of synthesized compounds.
Chemistry
[
a]
[b]
[c]
Compd
CC50 [mm]
EC50 [mm]
SI
All reactions were carried out in oven-dried glassware under nitro-
gen atmosphere. The chemicals and solvents were purchased from
Spectrochem, Across, Rankem, or Sigma–Aldrich. d-Ribose was pur-
chased from Carbosynth Limited. Melting points were recorded on
a Veego melting point apparatus. Analytical thin-layer chromatog-
6
6
6
6
a
b
c
>100
>100
80.7
43.6
>10
>100
87.6
32.2
6.6
–
–
2.5
6.6
58.8
d
[
d]
KZ-16
0.17
raphy (TLC) was performed on pre-coated plates (silica gel 60 F254
)
[
a] 50% cytotoxic concentration, determined from the decrease in viable
purchased from Merck. Purification by gravity column chromatog-
raphy was carried out on silica gel (100–200 mesh). An Elico UV/Vis
spectrophotometer was used for recording UV spectra. Optical ro-
tations of optically active compounds were measured on a Rudolph
cell counts. [b] 50% effective concentration, determined from inhibition
in luciferase activity. [c] CC50/EC50. [d] Phenanthridinone derivative.
[
23]
1
13
À1
Autopol I instrument. H and C NMR spectra were obtained on
quent structures was 0.05 kJmol . The initial conformations for all
molecules were obtained by 5000 steps of Monte Carlo conforma-
tional search (CS) under the GB/SA continuum water solvation
model using MMFFs force field and subsequent EM by the Polak–
Ribiere conjugate gradient (PRCG) method. The top-five conforma-
tions were selected for conformational studies. The online server
PROSIT was used to calculate geometrical parameters, including
the pseudo-rotation angle of NTMs. The lowest-energy conformer
of each NTM was submitted for analysis of its drug-like properties
using the QikProp module of Schrçdinger.
Varian or Bruker spectrometers (400 and 100 MHz) using CDCl or
3
[
D ]DMSO as solvents. Peaks are recorded with the following ab-
6
breviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; J, coupling constant (Hz).
General procedure for the synthesis of 6a–d
DIAD (3.75 mmol) was added dropwise to a mixture of the appro-
priate compound 3a–d (1.5 mmol), key cyclopentene intermediate
1
(1.57 mmol), and Ph P (3.75 mmol) in THF at 08C. The reaction
[
8]
3
The HCV RdRp model was built as previously described and re-
fined based on the crystal structure of HCV RdRp complex (PDB ID:
4
mixture was brought to RT, and stirring continued. Reaction com-
pletion was analyzed by TLC, the solvent was evaporated under re-
duced pressure, and the crude was purified by column chromatog-
raphy on silica gel by eluting with up to 30% EtOAc in hexane to
give coupling products 5a–d in 70–80% yields.
[13]
E7A). ATP was docked into the nucleoside active site to gener-
ate the HCV RdRp–ATP model. The ATP-docked model was further
subjected to 5 ns molecular dynamics (MD) simulation using Des-
mond, and the average structure was chosen. The final HCV RdRp
model consists of the template–primer complex (2:3), the natural
ATP substrate, along with the two Mg ions. It was further opti-
mized and checked by the Protein Preparation module and EM by
Macromodel using the OPLS2005 force field for 5000 steps with
the GB/SA continuum water solvation model. The lowest-energy
conformer of triphosphate analogues of 6a–d (NTMs) were select-
The deprotection of trityl, acetonide, and Boc groups of 5a–d was
carried out by heating at 608C in 10% HCl in MeOH. After comple-
tion (monitored by TLC), the reaction mixture was concentrated
under reduced pressure, and the solid was washed with acetone
to yield pure HCl salts of carbocyclic nucleosides 6a–d in 70–85%
yield.
2
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Di-Boc-protected 1-((4R)-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-
dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (5a): Yield: 80%; mp: 818C; HRMS (m/z):
(1S,2R,5R)-5-(4-Amino-3-bromo-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6c): Yield: 80%;
+
+
mp: 189–1908C; MS (ESI) (m/z): [M ] 342.0, [M +2] 344.0; UV
+
1
21
3
À1
À1
7
68.7283 [M +Na]; UV (MeOH): lmax =265.7 nm; H NMR
(MeOH): lmax =262 nm; [a]D =À155.62 cm g dm
(c=0.18
1
(
400 MHz, CDCl ): d=8.76 (s, 1H, 2-CH), 8.13 (s, 1H, 7-CH), 7.20–
MeOH); H NMR (400 MHz, [D ]DMSO): d=9.18–9.25 (bs, 1H, NH ),
3
6
2
7
5
.45 (m, 15H, trityl), 6.05–6.10 (m, 2H, 1’,6’-CH), 5.33–5.35 (d, J=
.5 Hz, 1H, 2’-CH), 4.87–4.88 (d, J=5.5 Hz, 1H, 3’-CH), 3.95–3.98 (d,
8.47 (s, 1H, 2-CH), 8.18–8.28 (bs, 1H, NH ), 5.66–5.67 (m, 1H, 1’-CH),
2
5.56–5.57 (m, 1H, 6’-CH), 4.45–5.22 (bs, 3H, 2’, 3’ & 5’-OH), 4.37–
4.39 (m, 1H, 2’-CH), 4.24–4.27 (m, 1H, 3’-CH), 4.06–4.14 ppm (m,
J=14.8 Hz, 1H, 5’-CH ), 3.78–3.82 (d, J=14.8 Hz, 1H, 5’-CH ), 1.55
2
2
13
(
s, 18H, Boc-6CH ), 1.45 (s, 3H, CH ), 1.33 ppm (s, 3H, CH ).
2H, 5’-CH2); C NMR (100 MHz, [D ]DMSO): d=153.48, 153.02,
3
3
3
6
1
5
50.97, 150.54, 123.53, 120.16, 99.77, 76.92, 72.39, 67.57,
8.93 ppm.
Di-Boc-protected
methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (5b): Yield: 83%; mp: 70–748C;
3-chloro-1-((4R)-2,2-dimethyl-6-((trityloxy)-
(1S,2R,5R)-5-(4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-
+
+
3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6d): Yield: 81%; mp:
194–1968C;
MS (ESI) (m/z): [M À100 (Boc)] 680.0, [M +2À100] 682.0; UV
+
21
1
MS
(ESI)
(m/z):
[M +1]
390.0;
[a]D
=
(
MeOH): l =263.0 nm; H NMR (400 MHz, CDCl ): d=8.96 (s, 1H,
max
3
3
À1
À1
À147.88 cm g dm (c=0.24 MeOH); UV (MeOH): lmax =264 nm;
2
5
-CH), 7.47–7.22 (m, 15H, trityl), 6.05–6.09 (m, 2H, 1’,6’-CH), 5.33–
.34 (d, J=5.7 Hz, 1H, 2’-CH), 4.87–4.88 (d, J=5.7 Hz, 1H, 3’-CH),
1
H NMR (400 MHz, [D ]DMSO): d=9.18–9.22 (bs, 1H, NH ), 8.49 (s,
6
2
1
5
1
H, 2-CH), 8.02–8.21 (bs, 1H, NH ), 5.64–5.65 (m, 1H, 1’-CH), 5.56–
3
.96–4.00 (d, J=15.4 Hz, 1H, 5’-CH ), 3.81–3.84 (d, J=15.4 Hz, 1H,
2
2
.57 (m, 1H, 6’-CH), 4.61–5.22 (bs, 3H, 2’, 3’ & 5’-OH), 4.38–4.39 (m,
H, 2’-CH), 4.26–4.29 (m, 1H, 3’-CH), 4.07–4.11 ppm (m, 2H, 5’-
5
’-CH ), 1.49 (s, 3H, CH ), 1.47 (s, 18H, Boc-6CH ), 1.44 ppm (s, 3H,
2
3
3
CH3).
13
CH2); C NMR (100 MHz, [D ]DMSO): d=153.06, 152.10, 150.23,
6
Di-Boc-protected
3-bromo-1-((4R)-2,2-dimethyl-6-((trityloxy)-
148.93, 123.23, 102.68, 92.39, 76.45, 71.82, 67.00, 58.39 ppm.
methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (5c): Yield: 82%; mp: 85–878C;
+
+
MS (m/z): [M À100] 724.0, [M +2À100] 726.0; UV (MeOH): l
=
max
1
2
63 nm; H NMR (400 MHz, CDCl ): d=8.96 (s, 1H, 2-CH), 7.21–7.46
3
Anti-HCV activity
(
m, 15H, trityl) 6.04–6.08 (m, 2H, 1’,6’-CH), 5.32–5.34 (d, J=5.8 Hz,
1
1
1
H, 2’-CH), 4.87–4.88 (d, J=5.9 Hz, 1H, 3’-CH), 3.95–3.99 (d, J=
The anti-HCV activities of test compounds 6a–d were determined
[24]
5.3 Hz, 1H, 5’-CH ), 3.80–3.83 (d, J=15.3 Hz, 1H, 5’-CH ), 1.44 (s,
in LucNeo#2 cells by using the previously described method with
2
2
[25]
8H, Boc-6CH ), 1.42 (s, 3H, CH ), 1.33 ppm (s, 3H, CH ).
some modifications. All compounds were dissolved in DMSO at
0 mm or higher to exclude the cytotoxicity of DMSO and stored
3
3
3
2
Di-Boc-protected 3-iodo-1-((4R)-2,2-dimethyl-6-((trityloxy)meth-
yl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-1H-pyrazolo-
at À208C until use. Huh-7 cells containing self-replicating sub-ge-
nomic replicons with a luciferase reporter, LucNeo#2, were grown
and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with
[
3,4-d]pyrimidin-4-amine (5d): Yield: 85%; mp: 89–998C; MS (ESI)
+
1
(m/z): [M +1] 872.0; UV (MeOH): l =264 nm; H NMR (400 MHz,
high glucose (Gibco/BRL) supplemented with 10% heat-inactivated
max
À1
CDCl ): d=8.97 (s, 1H, 2-CH), 7.21–7.46 (m, 15H, trityl), 6.04–6.08
fetal bovine serum (Gibco/BRL), 100 UmL
penicillin G, and
3
À1
(
m, 2H, 1’,6’-CH), 5.33–5.35 (d, J=5.8 Hz, 1H, 2’-CH), 4.88–4.90 (d,
100 mgmL
streptomycin. LucNeo#2 cells were maintained in
DMEM containing 1 mgmL G418 (Nakarai Tesque). Briefly, cells
(5ꢂ10 cells per well) were cultured in a 96-well plate in the ab-
À1
J=5.9 Hz, 1H, 3’-CH), 3.95–3.99 (d, J=15.3 Hz, 1H, 5’-CH ), 3.80–
2
3
3
6
.84 (d, J=15.3 Hz, 1H, 5’-CH ), 1.45 (s, 3H, CH ), 1.42 (s, 18H, Boc-
2
3
CH ), 1.33 ppm (s, 3H, CH ).
sence of G418 and in the presence of various concentrations of
test compounds. After incubation at 378C for 3 days, the culture
medium was removed, and cells were washed twice with phos-
phate-buffered saline. Lysis buffer was added to each well, and the
lysate was transferred to the corresponding well of a non-transpar-
ent 96-well plate. Luciferase activity was measured after the addi-
tion of luciferase reagent in a luciferase assay system kit (Promega),
using a luminometer with automatic injectors (Berthold Technolo-
gies). The number of viable cells was determined by a dye-based
method using the water-soluble tetrazolium Tetracolor One (Seika-
gaku Corporation), according to the manufacturer’s instructions.
3
3
(1S,2R,5R)-5-(4-Amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(hy-
droxymethyl)cyclopent-3-ene-1,2-diol (6a): Yield: 60%; mp: 189–
+
21
1
928C;
HRMS
(m/z):
286.1638
[M +Na];
[a]D
=
3
À1
À1
À120.29 cm g dm (c=0.38 MeOH); UV (MeOH): lmax =264 nm;
1
H NMR (400 MHz, [D ]DMSO): d=9.71 (bs, 1H, NH ), 8.83 (bs, 1H,
6
2
NH ), 8.45 (s, 2H, 2-CH & 7-CH), 5.66–5.67 (m, 1H, 1’-CH), 5.56–5.57
2
(
m, 1H, 6’-CH), 4.41–4.42 (m, 1H, 2’-CH), 4.29–4.32 (m, 1H, 3’-CH),
4
.06–4.15 (m, 2H, 5’-CH ), 3.16–3.65 ppm (bs, 3H, 2’, 3’ & 5’-OH);
2
1
3
C NMR (100 MHz, [D ]DMSO): d=153.09, 151.55, 150.50, 148.57,
6
1
35.31, 123.86, 100.01, 76.86, 72.53, 67.15, 58.92 ppm.
(1S,2R,5R)-5-(4-Amino-3-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6b): Yield: 71%;
+
+
mp: 206–2088C; MS (ESI) (m/z): [M ] 297.8, [M +2] 299.8; UV Acknowledgements
2
1
3
À1
À1
(
MeOH): lmax =267 nm; [a]D =À164.04 cm g dm
(c=0.15
1
MeOH); H NMR (400 MHz, [D ]DMSO): d=9.40 (bs, 1H, NH ), 8.73
We acknowledge financial support from the Department of Sci-
ence & Technology, Delhi (India) through grant no. SR/FT/CS-001/
6
2
(
5
1
bs, 1H, NH ), 8.50 (s, 1H, 2-CH), 5.84–6.21 (bs, 3H, 2’, 3’ & 5’-OH),
.66–5.67 (m, 1H, 1’-CH), 5.57–5.58 (m, 1H, 6’-CH), 4.38–4.40 (m,
2
2009. M.K. and T.B. thank CSIR (India) for the award of a Senior
H, 2’-CH), 4.24–4.27 (m, 1H, 3’-CH), 4.06–4.15 ppm (m, 2H, 5’-
Research Fellowship (SRF). A.S. thanks the Department of Bio-
technology, India for modeling software support. The authors ac-
knowledge Dr. Reddy’s Institute of Life Sciences, Hyderabad for
use of the NMR–Mass Spectrometry Facility, and the Central In-
strument Facility at BIT Mesra for analytical support.
1
CH ); H NMR (400 MHz, [D ]DMSO, D O exchange): d=8.29 (s, 1H,
2
4
(
1
2
6
2
-CH), 5.62–5.65 (m, 2H, 1’ & 6’-CH), 4.40–4.41 (m, 1H, 2’-CH), 4.20–
13
.23 (m, 1H, 3’-CH), 4.08–4.10 ppm (m, 2H, 5’-CH2); C NMR
100 MHz, [D ]DMSO): d=152.76, 152.71, 151.13, 150.00, 133.38,
6
23.34, 97.55, 76.89, 72.42, 67.58, 58.93 ppm.
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Published online on && &&, 0000
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FULL PAPERS
A well-defined shape analysis is
M. Kasula, T. Balaraju, M. Toyama,
A. Thiyagarajan, C. Bal, M. Baba,
A. Sharon*
needed for correlating conformation
with biological activity. Our nucleoside
mimetic design process involves confor-
mational cluster analysis to evaluate the
preferred “anti” conformation of the
base with respect to the 6-position hy-
drogen bond donor and 2’-exo confor-
mations for anti-HCV activity. In nepla-
nocin A, the adenine base shows 80%
syn with only 20% in the preferred anti
conformation in carbocyclic nucleosides.
&& – &&
A Conformational Mimetic Approach
for the Synthesis of Carbocyclic
Nucleosides as Anti-HCV Leads
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2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers!