Regioselective Synthesis of Pyrazolo[3,4-D[Pyrimidine Based Carbocyclic Nucleosides as Possible Antiviral Agent

Article abstract of DOI:10.1080/15257770.2015.1114126

Carbocyclic nucleosides are considered as nucleoside mimetic having high therapeutic potentials, however diverse exploration is still limited due to their synthetic difficulties. The major challenges are associated with the preparation of new base and carbocyclic sugar key intermediates. The modified base may provide conformational advantage to achieve better nucleoside mimetics and may also help in increasing the drug-like properties. In this manuscript, we report the use of acetamidine hydrochloride to synthesize 6-methyl-4-amino-pyrazolo[3,4-d]pyrimidine base and regioselective synthesis of six new carbocyclic nucleosides (6a-f) for antiviral evaluation. Theoretical investigations were carried out on the basis of thermodynamic and kinetic stability using MM based energy optimizations and QM based transition state search for the significant regioselectivity, which was further experimentally analyzed by NOE and UV spectroscopy.

Full text of DOI:10.1080/15257770.2015.1114126

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Regioselective Synthesis of Pyrazolo[3,4-
D]Pyrimidine Based Carbocyclic Nucleosides as
Possible Antiviral Agent
Mohan Kasula, Ramakrishnamraju Samunuri, Harapriya Chakravarty,
Chandralata Bal, Masanori Baba, Ashok Kumar Jha & Ashoke Sharon
To cite this article: Mohan Kasula, Ramakrishnamraju Samunuri, Harapriya Chakravarty,
Chandralata Bal, Masanori Baba, Ashok Kumar Jha & Ashoke Sharon (2016): Regioselective
Synthesis of Pyrazolo[3,4-D]Pyrimidine Based Carbocyclic Nucleosides as Possible Antiviral
Agent, Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2015.1114126
To link to this article: http://dx.doi.org/10.1080/15257770.2015.1114126
Published online: 25 Jan 2016.
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Date: 02 February 2016, At: 04:22

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
http://dx.doi.org/./..
Regioselective Synthesis of Pyrazolo[,-D]Pyrimidine
Based Carbocyclic Nucleosides as Possible Antiviral Agent
Mohan Kasulaa, Ramakrishnamraju Samunurib, Harapriya Chakravartya,
Chandralata Bala, Masanori Babac, Ashok Kumar Jhab, and Ashoke Sharona
aDepartment of Chemistry, Birla Institute of Technology, Mesra, Ranchi, India; bChemistry Services, GVK
Biosciences Pvt. Ltd., IDA Uppal, Hyderabad, India; cDivision of Antiviral Chemotherapy, Center for Chronic
Viral Diseases, Kagoshima University, Kagoshima, Japan
ABSTRACT
ARTICLE HISTORY
Received  April 
Accepted  October 
Carbocyclic nucleosides are considered as nucleoside mimetic
having high therapeutic potentials, however diverse exploration
is still limited due to their synthetic difficulties. The major chal-
lenges are associated with the preparation of new base and car-
bocyclic sugar key intermediates. The modified base may provide
conformational advantage to achieve better nucleoside mimet-
ics and may also help in increasing the drug-like properties. In
this manuscript, we report the use of acetamidine hydrochloride
to synthesize 6-methyl-4-amino-pyrazolo[3,4-d]pyrimidine base
and regioselective synthesis of six new carbocyclic nucleosides
(6a-f) for antiviral evaluation. Theoretical investigations were car-
ried out on the basis of thermodynamic and kinetic stability
using MM based energy optimizations and QM based transition
state search for the significant regioselectivity, which was further
experimentally analyzed by NOE and UV spectroscopy.
KEYWORDS
Mitsunobu reaction;
neplanocin; conformation;
nucleoside mimetic; antiviral
agent
Abbreviations: 4-APP; 4-amino-pyrazolo[3,4-d]pyrimidine; 6-methyl-4-APP; 6-
methyl-4-amino-pyrazolo[3,4-d]pyrimidine; HCV; Hepatitis C Virus
1. Introduction
Carbocyclic nucleosides are nucleoside analogs in which -O- of the furanoside is
replaced by its bioisostere -CH2- to generate a carbocyclic moiety which has an
increased metabolic stability due to an improvement in resistance against the enzy-
matic hydrolysis[1] and a ring-puckering which may be useful to exhibit new biolog-
ical properties.[2] Hence, these molecules may serve as better nucleoside mimic for
competitive inhibiton of viral polymerase.[3] It is important to note that the natu-
rally occuring carbocyclic nucleosides aristeromycin[4] and neplanocin A[5] exhibits
significant antivral activities[6] but were found to be toxic.[7,8] In addition, abacavir
CONTACT Ashoke Sharon
Mesra, Ranchi-, India.
asharon@bitmesra.ac.in
Department of Chemistry, Birla Institute of Technology,
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

2
MOHAN KASULA ET AL.
Figure . The chemical structure of aristeromycin, neplanocin A, abacavir (approved anti-HIV
drug), entecavir (approved anti-HBV drug) and target molecules based on -methyl--amino-
H-pyrazolo[,-d] pyrimidine based carbocyclic nucleosides (I) for synthesis and biological
evaluation.
(approved anti-HIV drug), entecavir (approved anti-HBV drug) belongs to this class
of nucleoside which provides us a vision to explore novel carbocyclic nucleosides as
possible antivirals.
Recently, we have synthesized and explored less toxic neplanocin based analogs
with 4-amino-1H-pyrazolo[3,4-d]pyrimidine (4-APP) base having some antiviral
activity.[9] Therefore, in continuation to our previous work, we chose to synthesize
a new class of more hydrophobic carbocyclic analogs based on 6-methyl-4-amino-
1H-pyrazolo[3,4-d]pyrimidine (6-methyl-4-APP, I, Figure 1) as possible antiviral
agents. Synthesis of this base was earlier reported[10] starting from 3-amino-4-cyano
pyrazole with relatively harsh conditions and low yield (∼30%). Here, we present a
new high yield synthesis of 6-methyl-4-APP followed by regioselective synthesis of
new carbocyclic nucleosides I.
2. Results and discussion
2.1. Chemistry
The carbocyclic sugar key intermediate (1) as a single isomer was prepared start-
ing from the commercially available D-ribose in eight consecutive steps.[11,12] The
target base 3 was synthesized starting from malononitrile followed by cyclization
of 2 with acetamidine hydrochloride (Scheme 1). According to our knowledge this
is the first report to use acetamidine hydrochloride as an imidine precursor to
construct the pyrimidine ring. In brief, acetamidine hydrochloride converts into
acetamidine upon dissolving in ethanolic solution of sodium ethoxide. The imidine
group of acetamidine act as a nucleophile to attack the carbon atom of the cyano
group followed by cyclization with the release of ammonia giving 3 in 82% yield
(Scheme 1).

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme . Reagents and Condition: (i) Acetamidine hydrochloride, NaOEt, EtOH, °C,  h.
Reaction of 3 with di-tert-butyl dicarbonate [(Boc)2O] in presence of DMAP as
catalyst in THF followed by treatment with aq. NaHCO3 in methanol[13] yielded
the di-Boc protected base 4a (Scheme 2). The di-Boc derivatives of 3-halo analogs
(4b-d) were synthesized by treating 3 with respective N-halosuccinimide in DMF
followed by reaction with di-tert-butyl dicarbonate [(Boc)2O] in presence of DMAP
and with aq. NaHCO3 in methanol.
Mitsunobu coupling of 4a-d with 1 yielded regio-selective products 5a-d
(Scheme 3). The UV spectra of 5a-d were close to their respective Boc protected
aglycons (4a-d), which indicates only N1 glycosylation.[9,14] To further support the
selectivity, we performed NOE experiment. According to Seela et al. the irradia-
tion of H3 of the N2 glycosylated product should exhibit NOEs with sugar ring
protons.[15] In our case, the irradiation of H3 (on base moiety) of 5a showed nocor-
relation with any sugar ring protons (in Supporting information).
To further support our assignment, we performed the conformational search
analysis for N1 and N2 isomers that could be theoretically formed during the cou-
pling of 4a with 1. We found that the distances between H3 and sugar ring pro-
tons are in the NOE range for N2 isomer while they are out of NOE range in
case of N1. The rate determining step for the coupling of 4a with 1 was studied
by means of computational calculations and reaction mechanism (Figure 2) before
assigning and understanding the selectivity for N1 isomers (5a-d) (discussed in
2.2). These experimental data provides strong evidences for the formation of N1
isomer.
Scheme . Reagents and conditions: (i) (Boc)O, DMAP, THF, rt,  h (ii) aq. NaHCO, MeOH, rt,  h
(iii) NCS/NBS/NIS, DMF, rt,  h.

4
MOHAN KASULA ET AL.
Scheme . Synthesis of -methy--APP based carbocyclic nucleoside analogs 6a-e.
Reagents and conditions: (i) Ph3P, DIAD, THF, 0–5°C, 2 h; (ii) Methanolic HCl,
rt, 5–10 h; (iii) Tributyl(vinyl) stannane, Pd(PPh3)4, CuI, Triethyl amine, 110°C, 5 h;
(iv) 3-butyn-1-ol, Pd(PPh3)4, CuI, Triethyl amine, 110°C, 10 h.
The deprotection of 5a-d was carried out by treating them in methanolic HCl
to yield the desired carbocyclic nucleosides (6a-d). Stille and Sonogashira coupling
reactions were utilized for the synthesis of 6e and 6f from 6d, respectively (Scheme
3). In brief, aryl halide 6d was treated with tributylvinyltin and homopropargyl alco-
hol to introduce the vinyl and the homopropargyl alcohol groups, respectively.
Figure . The plausible mechanism for Mitsunobu coupling of 4a with 1.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
Figure . MM energy analyses showing the N-regioisomer to have lower energy than N-isomer.
2.2. Computational studies
To investigate the regioselectivity of Mitsunobu coupling, energetic investigation
was performed. The molecular mechanics (MM) energy optimization indicated that
the potential energy of the N1 isomer is lower than the corresponding N2 as shown
in Figure 3. This suggests that the selective formation of N1 isomer is due to its
higher stability with respect to N2.
The quantum mechanics (QM) calculation for the activation energy for the for-
mation of 5a (N1) and 5a (N2) were carried out. The result shows that the transition
state for N1 is 0.0016 Ha (4.27 kJ/mol) lower in energy than that of N2 (schematic
representation in Figure 4). In addition, the relative energy of N1 isomer is less than
that of N2 isomer. Thus, QM calculation also supports the formation of N1 product.
In contrast, when Yang et al. performed the Mitsunobu coupling of the same sugar
Figure . Schematic representation of the energy barrier for the formation of N and N isomer.

6
MOHAN KASULA ET AL.
with a 4-chloro substituted base, obtained both the N1 and N2 isomers in a ratio
of 42:53.[16] It implies that the steric crowding at 4-postion (bulky Boc groups on
nitrogen) reduces nucleophilicity of N2 base and raises the activation energy for the
formation of N2 isomer. Furthermore, the steric hinderence between the bulky OTr
and N(Boc)2 groups destabilizes the N2 isomer (Figure 2). Therefore, the absolute
regioselectivity can be accounted to the fact that the N1 is not only thermodynami-
cally stable but also kinetically favored.
3. Experimental
3.1. General methods
All reactions. were carried out in oven-dried glassware under nitrogen atmosphere.
The chemicals and solvents were purchased from Spectrochem, Acros, Rankem or
Sigma-Aldrich. 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 chro-
matography was carried out on silica gel (100–200 mesh). Eleco UV/Vis spectropho-
tometer was used for recording the UV spectra. 1H/13C NMR were obtained from a
Varian (400 MHz) spectrometer or Bruker spectrometer using CDCl3 or DMSO-d6,
as solvents. Peaks are recorded with the following abbreviations: s, singlet; bs, broad
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling constant (hertz).
3.2. General procedure for the synthesis of 6a-d
To a mixture of appropriate 4a-d (1.57 mmol), sugar key intermediate 1 (1.50 mmol)
and Ph3P (3.75 mmol) in THF was added DIAD (3.75 mmol) dropwise at 0°C. The
reaction mixture was thereafter brought up to rt and stirring was continued. Com-
pletion of the reaction was analyzed by TLC, 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 (5a-d). The trityl,
acetonide and Boc groups of 5a-d were deprotected by stirring at rt in methano-
lic HCl. After completion (monitored by TLC), the reaction mixture was concen-
trated under reduced pressure and solid was dissolved in methanol; neutralized with
NaHCO3 and the crude was purified by silica gel column chromatography using
dichloromethane/methanol (5–10%) to yield pure carbocyclic nucleosides 6a-d. To
a solution of 6d (1.5 mmol) in dry DMF, tributylvinyltin (3.1 mmol), Pd(PPh3)4
(0.07 mmol) were added and stirred at 110°C for 5 h to obtaine 6e. Synthesis of 6f
was carried out using the same procedure wherein the homopropargyl alcohol was
added instead of tributylvinyltin, CuI (0.3 mmol), triethylamine (7.5 mmol).
Di-Boc protected 1-((3aS,4R,6aR)-2,2-dimethyl-6-(trityloxymethyl)-4,6a-
dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-6-methyl-1H-pyrazolo[3,4-d]
pyrimidin-4-amine (5a) Yield: 70%; mp: 68–70°C; MS-ESI (m/z): 760.2 [M++1];
UV (MeOH): λ
214 nm, 270 nm; 1H NMR (400 MHz, CDCl3): δ 8.09 (s, 1H,
max

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
3-CH), 7.21–7.46 (m, 15H, Tr-H), 6.04–6.10 (m, 2H, 1ꢀ, 6ꢀ-CH), 5.36–5.37 (d, J3=
5.9 Hz, 1H, 2ꢀ-CH), 4.87–4.89 (d, J3= 5.7 Hz, 1H, 3ꢀ-CH), 3.95–3.99 (d, J2= 14.9 Hz,
1H, 5ꢀ-CH), 3.77–3.81 (d, J2= 15.4 Hz, 1H, 5ꢀ-CH), 2.71 (s, 3H, 6-CH3), 1.55 (s,
18H, Boc-6CH3), 1.45 (s, 3H, CH3), 1.34 (s, 3H, CH3).
Di-Boc protected 3-chloro-1-((3aS,4R,6aR)-2,2-dimethyl-6-(trityloxymet-
hyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-6-methyl-1H-
pyrazolo[3,4-d]pyrimidin-4-amine (5b) Yield: 80%; mp: 70–74°C; MS-ESI
(m/z): [M+-100] 694.2, [M++2–100] 696.2; UV (MeOH) λmax: 215.0, 271.0 nm;
1H NMR (400 MHz, CDCl3): 7.21–7.46 (m, 15H, Tr-H), 6.02–6.07 (m, 2H, 1ꢀ,
6ꢀ-CH), 5.32–5.34 (d, J= 5.3 Hz, 1H, 2ꢀ-CH), 4.85–4.86 (d, J3= 5.7 Hz, 1H, 3ꢀ-CH),
3.94–3.98 (d, J2= 15.3 Hz, 1H, 5ꢀ-CH), 3.77–3.80 (d, J2= 15.3 Hz, 1H, 5ꢀ-CH), 2.81
(s, 3H, 6-CH3), 1.46 (s, 18H, Boc-6CH3), 1.45 (s, 3H, CH3), 1.33 (s, 3H, CH3).
Di-Boc protected 3-bromo-1-((3aS,4R,6aR)-2,2-dimethyl-6-(trityloxymeth-
yl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-6-methyl-1H-pyrazolo
[3,4-d]pyrimidin-4-amine (5c) Yield: 77%; mp: 71–72°C; MS (m/z): [M+] 837.8,
[M++2] 839.8; UV (MeOH) λ max: 216.9, 269.4nm; 1H NMR (400 MHz, CDCl3):
7.21–7.46 (m, 15H, Tr-H) 6.02–6.07 (m, 2H, 1ꢀ, 6ꢀ-CH), 5.33–5.35 (d, J3= 5.7 Hz,
1H, 2ꢀ-CH), 4.87–4.88 (d, J3= 5.7 Hz, 1H, 3ꢀ-CH), 3.95–3.98 (d, J2= 15.3 Hz, 1H,
5ꢀ-CH), 3.78–3.82 (d, J2= 15.3 Hz, 1H, 5ꢀ-CH), 2.82 (s, 3H, 6-CH3), 1.45 (s, 18H,
Boc-6CH3), 1.43 (s, 3H, CH3), 1.33 (s, 3H, CH3).
Di-Boc protected 3-iodo-1-((3aS,4R,6aR)-2,2-dimethyl-6-(trityloxymethyl)-
4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-6-methyl-1H-pyrazolo[3,4-
d]pyrimidin-4-amine (5d) Yield: 73%; mp: 74–75 °C; MS-ESI (m/z): [M+] 885.7;
UV (MeOH) λ max: 210.2, 269.3 nm; 1H NMR (400 MHz, CDCl3): 7.21–7.47 (m,
15H, Tr-H), 6.02–6.06 (m, 2H, 1ꢀ, 6ꢀ-CH), 5.34–5.36 (d, J3= 5.3 Hz, 1H, 2ꢀ-CH),
4.88–4.90 (d, J3= 5.5 Hz, 1H, 3ꢀ-CH), 3.94–3.98 (d, J2= 15.4 Hz, 1H, 5ꢀ-CH), 3.78–
3.82 (d, J2= 14.9 Hz, 1H, 5ꢀ-CH), 2.82 (s, 3H, 6-CH3), 1.44 (s, 18H, Boc-6CH3),
1.39 (s, 3H, CH3), 1.33 (s, 3H, CH3).
(1S,2R,5R)-5-(4-amino-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-
(hydroxymethyl) cyclopent-3-ene-1,2-diol (6a) Yield: 80%, mp > 250°C; HR-MS
19
(m/z): [M++1] 278.0670; [α]D −26.39 (c 0.082 MeOH); UV (MeOH) λ max 215,
1
270.2 nm; H NMR (400 MHz, DMSO-d6) δ 10.06 (bs, 1H, NH), 8.64 (bs, 1H,
NH), 8.49 (s, 1H, 3-CH), 5.65–5.66 (m, 1H, 1ꢀ-CH), 5.53–5.54 (m, 1H, 6ꢀ-CH),
4.40–4.42 (m, 1H, 2ꢀ-CH), 4.29–4.31 (m, 1H, 3ꢀ-CH), 4.10–4.13 (m, 2H, 5ꢀ-CH),
1
3.16–3.65 (bs, 3H, 3-OH), 2.57 (s, 3H, 6-CH3); H NMR (400 MHz, DMSO-d6,
D2O exchange) δ 8.32 (s, 1H, 3-CH), 5.62–5.63 (m, 1H, 1ꢀ-CH), 5.54–5.55 (m, 1H,
6ꢀ-CH), 4.39–4.40 (m, 1H, 2ꢀ-CH), 4.26–4.29 (m, 1H, 6ꢀ-CH), 4.08–4.10 (m, 2H,
5ꢀ-CH), 2.51 (s, 3H, 6-CH3).
(1S,2R,5R)-5-(4-amino-3-chloro-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6b) Yield: 78%; mp: >250°C;
MS-ESI (m/z): [M+] 311.6, [M++2] 313.6; UV (MeOH) λ
215.0, 278.0nm;
max
20
[α]D −70.22 (c 0.08 MeOH); 1H NMR (400 MHz, DMSO-d6) δ 7.8–8.2 (bs, 1H,
NH), 6.8–7.2 (bs, 1H, NH), 5.59–5.60 (m, 1H, 1ꢀ-CH), 5.50–5.51 (m, 1H, 6ꢀ-CH),
4.90–5.04 (m, 3H, 3-OH), 4.33–4.36 (m, 1H, 2ꢀ-CH), 4.19–4.24 (m, 1H, 3ꢀ-CH),

8
MOHAN KASULA ET AL.
1
4.05–4.06 (m, 2H, 5ꢀ-CH), 2.37 (s, 3H, 6-CH3); H NMR (400 MHz, DMSO-d6,
D2O exchange) 5.58–5.63 (m, 2H, 1ꢀ, 6ꢀ-CH), 4.41–4.42 (m, 1H, 2ꢀ-CH), 4.24–4.27
(m, 1H, 3ꢀ-CH), 4.10–4.13 (m, 2H, 5ꢀ-CH), 2.42 (s, 3H, 6-CH3).
(1S,2R,5R)−5-(4-amino-3-bromo-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-
1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6c) Yield: 75%; mp: >250°C;
26
MS-ESI (m/z): [M+] 355.5, [M++2] 357.5; [α]D −42.23 (c 0.092 MeOH); UV
1
(MeOH) λ
215.7nm, 279.4nm; H NMR (400 MHz, DMSO-d6) δ 7.7–8.2
max
(bs, 1H, NH), 6.6–7.2 (bs, 1H, NH), 5.62–5.63 (m, 1H, 1ꢀ-CH), 5.52–5.53 (m,
1H, 6ꢀ-CH), 4.93–5.06 (m, 3H, 3-OH), 4.33–4.36 (m, 1H, 2ꢀ-CH), 4.23–4.26 (m,
1H, 3ꢀ-CH), 4.07–4.08 (m, 2H, 5ꢀ-CH), 2.40 (s, 3H, 6-CH3); 1H NMR (400 MHz,
DMSO-d6, D2O exchange) 5.63–5.64 (m, 1H, 1ꢀ-CH), 5.58–5.59 (m, 1H, 6ꢀ-CH),
4.40–4.42 (m, 1H, 2ꢀ-CH), 4.25–4.28 (m, 1H, 3ꢀ-CH), 4.12–4.13 (m, 2H, 5ꢀ-CH),
2.43 (s, 3H, 6-CH3).
(1S,2R,5R)-5-(4-amino-3-Iodo-6-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6d) Yield: 71%; mp: >250°C;
24
HR-MS-ESI (m/z): [M+] 403.9974; [α]D −99.07 (c 0.18 MeOH); UV (MeOH)
λ
215.7nm, 282.9nm; 1H NMR (400 MHz, DMSO-d6) δ 7.2–7.8 (bs, 1H, NH),
max
6.2–6.8 (bs, 1H, NH), 5.60–5.61 (m, 1H, 1ꢀ-CH), 5.51–5.52 (m, 1H, 6ꢀ-CH3),
4.94–5.06 (m, 3H, 3-OH), 4.36–4.38 (m, 1H, 2ꢀ-CH), 4.25–4.30 (m, 1H, 3ꢀ-CH3),
4.08–4.10 (m, 2H, 5ꢀ-CH), 2.40 (s, 3H, 6-CH3); H NMR (400 MHz, DMSO-d6,
1
D2O exchange) 5.59–5.63 (m, 2H, 1ꢀ, 6ꢀ-CH), 4.41–4.43 (m, 1H, 2ꢀ-CH), 4.28–
4.30 (m, 1H, 3ꢀ-CH), 4.09–4.13 (m, 2H, 5ꢀ-CH), 2.43 (s, 3H, 6-CH3); 13C NMR
(100 MHz, DMSO-d6) δ 165.52, 157.74, 155.39, 149.89, 124.80, 101.88, 89.89,
76.75, 72.39, 66.23, 58.93, 26.03.
(1S,2R,5R)-5-(4-amino-6-methyl-3-vinyl-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6e) Yield: 41%; mp: >250°C;
24
HR-MS-ESI (m/z): [M++1] 304.0576; [α]D −82.07 (c 0.12 MeOH); UV (MeOH)
1
λ
max
214.7nm, 284.9nm; H NMR (400 MHz, DMSO-d6) δ 7.20–7.27 (dd, 1H,
CH), 5.93–5.98 (t, 1H, CH), 5.68–5.69 (m, 1H, 1ꢀ-CH), 5.543–5.546 (d, 1H, 6ꢀ-CH),
5.34–5.37 (d, 1H, CH), 4.37–4.40 (q, 2H, 2ꢀ & 3ꢀ-CH), 4.10 (m, 2H, 5ꢀ-CH), 2.39 (s,
3H, 2-CH3); 1H NMR (400 MHz, DMSO-d6, D2O exchange) δ 7.16–7.23 (dd, 1H,
CH), 5.95–5.99 (d, 1H, CH), 5.69–5.71 (m, 1H, 1ꢀ-CH), 5.58–5.59 (m, 1H, 6ꢀ-CH),
5.42–5.45 (d, 1H, CH), 4.36–4.43 (m, 2H, 2ꢀ & 3ꢀ-CH), 4.12 (m, 2H, 5ꢀ-CH), 2.41
(s, 3H, 6-CH3); 13C NMR (100 MHz, DMSO-d6) δ 164.82, 158.20, 156.16, 149.43,
141.18, 128.17, 125.23, 117.59, 96.52, 76.59, 72.49, 65.60, 58.99, 25.97.
(1S,2R,5R)-5-(4-amino-3-(4-hydroxybut-1-ynyl)-6-methyl-1H-pyrazolo[3,4-
d]pyrimidin-1-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (6f) Yield: 30%;
20
mp: >250°C; MS-ESI (m/z): [M++1] 346.18; [α]D −79.06 (c 0.11 MeOH); UV
1
(MeOH) λ
210.5 nm, 279.3 nm; H NMR (400 MHz, DMSO-d6) δ 6.56–6.58
max
(bs, 1H, OH), 5.45–5.49 (d, 1H, J= 15.6 Hz, 1ꢀ-CH), 5.30–5.32 (d, 1H, J= 6.4 Hz,
6ꢀ-CH), 5.06–5.08 (bs, 1H, OH), 4.71–4.75 (bs, 1H), 4.61–4.66 (dd, 1H, J= 15.6 Hz,
J= 5.6 Hz, 2ꢀ-CH), 4.02–4.13 (m, 3H, 5ꢀ & 3ꢀ-CH), 3.61–3.63 (t, 2H, CH2), 2.64–
1
2.66 (t, 2H, CH2), 2.41–2.43 (d, 3H, CH3) H NMR (400 MHz, DMSO-d6, D2O
exchange) 5.50–5.54 (d, 1H, J= 15.6 Hz, 1ꢀ-CH), 5.39–5.40 (d, 1H, J= 6.4 Hz,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
6ꢀ-CH), 4.70–4.73 (dd, 1H, J= 15.6 Hz, J= 5.6 Hz, 2ꢀ-CH), 4.02–4.20 (m, 3H, 5ꢀ &
3ꢀ-CH), 3.63–3.66 (t, 2H, CH2), 2.64–2.67 (t, 2H, CH2), 2.42–2.44 (d, 3H, CH3).
3.3. Computer aided computational studies
To study the absolute selectivity obtained in Mitsunobu reaction from coupling
of 4a-d with 1, the structures of N1 and N2 isomers that could be formed were
build using Maestro interface of Schrödinger. Preliminary energy optimizations
were carried out using classical mechanics. The structures were minimized using
Macromodel[17] with Optimized Potentials for Liquid Simulations (OPLS) 2005
force field. The minimized structures were subjected to conformational search with
OPLS 2005 force field using Polak-Ribiere Conjugate Gradient method with a max-
imum of 5000 iterations and convergence threshold of 0.05. Further, the reaction
kinetics study for the rate determing step was performed using Transition State
Search protocol of Jaguar Module[18] Schrodinger. The linear synchronous tran-
sit (LST) method was used to elucidate the energetic pathways from reactant(s) to
formation of product(s) through transition state. The phosphonium ion interme-
diate of the sugar and the deprotonated base were used as the reactants and the
corresponding N1 and N2 isomers with triphenyl phosphine oxide as products. The
calculations were performed using spin restricted DFT with B3LYP (Becke-3-Lee-
Yang-Parr) density functional and the 6–31-G∗∗ basis set.
4. Conclusions
The 6-methyl-4-APP based carbocyclic nucelosides (6a-f) were successfully pre-
pared for antiviral evaluation. The base (3) was prepared using acetamidine
hydrochloride as an imidine precursor for the preparation of various 4-N(Boc)2
bases (4a-d). The Boc group at 4-position resulted in exclusive regioselective glyco-
sylation during Mitsunobu reaction to yield only N1 products (5a-d). Regioselectiv-
ity was investigated both theoretically by studying thermodynamic and kinetic sta-
bility and experimentally by analyzing NOE and UV spectra. The final compounds
6a-f were evaluated against HCV in subgenome HCV RNA replicon cells containing
the luciferase gene. However none of the compounds was found to be significantly
active.
Acknowledgments
This research received funding from University Grant Commision (UGC), New Delhi, India
through F. No. 37–110/2009. MK is thankful to CSIR, India for Senior Research Fellowship.
AS acknowledges DST FIST support for NMR and Computational Chemistry Facility. Authors
acknowledge Dr. Reddy’s Institute of Life Sciences, Hyderabad for NMR-Mass Facility and Cen-
tral Instrument Facility, BIT Mesra for analytical support.

10
MOHAN KASULA ET AL.
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