Solid-phase parallel synthesis of 4-β-d-ribofuranosylpyrazolo[4,3-d] pyrimidine nucleosides

Article abstract of DOI:10.1080/15257770500269432

□ The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d] pyrimidine-5,7-(4H,6H)-dione

Full text of DOI:10.1080/15257770500269432

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Solid-phase Parallel Synthesis of 4-β-D-
Ribofuranosylpyrazolo[4,3-d]pyrimidine Nucleosides
Kanda S. Ramasamy a , Roberto B. Amador a , Qazi Habib a , Frank Rong a , Xiaogang Han a
David Y. Li a , Jingfan Huang a , Zhi Hong a & Haoyun An a
,
a R&D Division , Valeant Pharmaceuticals , Costa Mesa, California, USA
Published online: 16 Aug 2006.
To cite this article: Kanda S. Ramasamy , Roberto B. Amador , Qazi Habib , Frank Rong , Xiaogang Han , David Y. Li ,
Jingfan Huang , Zhi Hong & Haoyun An (2005) Solid-phase Parallel Synthesis of 4-β-D-Ribofuranosylpyrazolo[4,3-d]pyrimidine
Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 1947-1970, DOI: 10.1080/15257770500269432
To link to this article: http://dx.doi.org/10.1080/15257770500269432
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1947–1970, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500269432
SOLID-PHASE PARALLEL SYNTHESIS OF
4-β-D-RIBOFURANOSYLPYRAZOLO[4,3-d]PYRIMIDINE
NUCLEOSIDES
Kanda S. Ramasamy, Roberto B. Amador, Qazi Habib, Frank Rong, Xiaogang
ᮀ
Han, David Y. Li, Jingfan Huang, Zhi Hong, and Haoyun An
R&D Division,
Valeant Pharmaceuticals, Costa Mesa, California, USA
ᮀ
The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel
synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-
(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H,6H)-dione (5) with 1-O-acetyl-2,3,5-tri-
O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides
6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides
6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12
and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and
C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B
and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the
solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously,
and they were analyzed and characterized by high-throughput LC-MS.
Keywords Solid phase; Parallel synthesis; Pyrazolo[4,3-d]pyrimidine nucleosides
INTRODUCTION
During the last few years, combinatorial chemistry has been used to
generate huge numbers of heterocyclic compound libraries and identified
lead candidate for different targets using high throughput screening. Thus,
combinatorial synthesis of large diverse libraries of organic molecules and
high throughput screening technologies are playing a key role in drug
discovery.[1] If nucleosides could be made through combinatorial chemistry
approach, a large number of nucleoside analogues could be synthesized
In honor and celebration of the life and career of John A. Montgomery.
Received 19 January 2005; accepted 26 June 2005.
The authors thank Vesna Stoisavljevic, Maja Stojiljkovic, Sharon Kolander, and Eugene Chang for
their help. The authors also thank Dr. Stanley Lang for critical review of the article.
Address correspondence to Kanda Ramasamy, Valeant Pharmaceuticals, 3300 Hyland Ave., Costa
Mesa, CA 92626. Fax: (714)641-7222; E-mail: kramasamy@valeant.com
1947

1948
K. S. Ramasamy et al.
within months instead of decades, and large nucleoside libraries could be
developed. A combinatorial approach to nucleosides may also encourage
scientists to focus beyond the biological targets such as DNA or RNA
polymerases, IMP dehydrogenase, protein kinases, and adenosine receptors.
Thus, if a vast number of diverse nucleoside analogues could be created,
their use extend far beyond these previously recognized biological targets
and this would open a new avenue for the use of nucleoside analogues as
human therapeutics.
Although, solid-phase combinatorial methodology has been applied to
synthesize highly functionalized libraries of heterocyclic compounds[2] and
oligonucleotides,[3] it has not been exploited in any significant fashion to
the synthesis of nucleosides.[4,5] This is due to: (a) the glycosidic bond for-
mation between a sugar moiety and a nucleoside base can be achieved only
through selective reaction conditions; (b) the liability of the glycosidic bond
to varying types of acids and reaction conditions; (c) most of the reaction
conditions that are being used in combinatorial chemistry approaches may
not be suitable to generate nucleoside libraries; (d) nucleosides have many
reactive sites that need to be selectively protected. Recently, two methods[6,7]
appeared in the literature: one utilized carboxypolystyrene solid support[6]
and the other[7] used solid support containing 2ꢁ,3ꢁ-acetal linkages, which
requires 5% TFA at 50◦C deprotection condition. Thus, there is a growing
interest to develop new solid-phase parallel synthesis methodologies for the
production of nucleoside libraries using different solid supports coupled
with mild cleavage condition from the solid support.
Pyrazolopyrimidine ring system forms a integral part of naturally occur-
ring nucleoside antibiotics such as Formycin A and B[8] and medicinally
useful drug such as allopurinol.[9] In addition, nucleosides having the pyra-
zolopyrimidine skeleton act as inhibitors of purine nucleoside phosphoryl-
ase.[10] Furthermore, newly discovered novel pyrazolopyrimidine derivatives
have demonstrated promising antimicrobial activities against Gram-positive
bacteria.[11] These interesting biological properties coupled with our ongo-
ing effort[12] to develop solid-phase parallel synthesis methodologies and
mild cleavage conditions to nucleosides prompted us to focus on nucleo-
sides having the pyrazolopyrimidine ring system.
RESULTS AND DISCUSSION
First, a prototype scaffold A (Figure 1) was selected to identify the sites
for diversity and the linking position on the solid support. Of course, the
scaffold A has many sites for diversity and for the linkage to the solid support
resin. However, a number of factors were considered in order to develop
a solid-phase parallel synthesis methodology to a prototype nucleoside
scaffold A (Figure 1): (a) identify a suitable resin and point of attachment

Solid-phase Parallel Synthesis of Nucleosides
1949
FIGURE 1 Prototype scaffold A.
for optimal nucleoside modifications and cleavage conditions that should
not affect the glycosidic bond, (b) exploit the advantage of solid phase
synthesis, and (c) design a strategy that could afford high quality products
and are amenable to combinatorial library production. In order to explore
the applicability of solid-phase chemistry to pyrazolopyrimidine scaffold A,
the diversity profile was set to a minimum. Therefore, the objective was to
introduce one site of diversity at the C7 position on the heterocyclic portion
of the scaffold A. To avoid N1 or N2 isomer complications, the pyrazole
ring of scaffold A was alkylated and the isomers separated before adding
on to the solid support. Also, the C7 carbonyl group of scaffold A has to
be converted to an “SCH3” functionality so that nucleophilic substitution
reaction at C7 can be performed effectively. Since the 5ꢁ-hydroxyl group
of the ribose moiety can be selectively protected[13] with trityl chloride,
dimethoxytrityl chloride, or monomethoxytrityl chloride in the presence of
unprotected 2ꢁ- and 3ꢁ-hydroxyl groups, the belief is that the corresponding
resins can also be used to link nucleosides at the 5ꢁ-position of the sugar
moiety. Interestingly, the 2ꢁ, 3ꢁ-hydroxyl groups of the sugar moiety A was
kept open because these groups can be further functionalized to provide
dideoxy nucleosides if needed. In addition, these solid support resins are
stable enough to introduce diversification on the heterocyclic part, and
cleavable under mild acidic or neutral conditions. Even though resins such
as trityl (Trt), dimethoxytrityl (DMT) and monomethoxytrityl (MMT) can
satisfy the above requirements, the MMT polystyrene resin was preferred.
The reasoning was that MMT might be cleavable under neutral condition

1950
K. S. Ramasamy et al.
rather than other trityl resins which require acidic conditions and more
stable than DMT resin. Hence, the main strategy was to link the solid
support on the 5ꢁ-position of the ribose moiety and introduce diversity at
the C7 position of pyrazolopyrimidine nucleoside scaffold A by keeping a
fixed functionality either at N1- or at N2-positions. Furthermore, the solid-
phase parallel synthesis methodology was applied to produce high-quality
nucleosides in parallel by utilizing the ACT Vanguard semi-automated
synthesizer combined with our in-house data processing techniques, which
enables us to have a facile quality control during library production,
purification, and high throughput screening.
Prior to exploring the solid phase chemistry, the key nucleosides 6 and
7 were synthesized starting from commercially available pyrazole derivative
1 as shown in Scheme 1. Alkylation of 1 with methyl iodide in the presence
of sodium hydride provided a mixture of N1 and N2 alkylated pyrazoles
2. The alkylated intermediates 2 were heated with methanolic ammonia at
110◦C in a steel bomb to give the corresponding amide 3 quantitatively.
Reduction of the nitro group of 3 followed by cyclization of the intermediate
4 with urea at high temperature afforded a mixture of N1- and N2-(methyl)-
1H- and 2H-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (5) as greenish
yellow solid. Silylation of the pyrazolopyrimidine 5 was accomplished by
heating of 5 with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) in the presence
of a catalytic amount of ammonium sulfate in anhydrous pyridine for 16 h
under an Argon atmosphere. The silyl derivative was then condensed with
1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of trimethylsilyl
triflate (TMStriflate) in anhydrous acetonitrile at room temperature. A
clean reaction was observed and the reaction products (6 and 7) were
separated by flash chromatography over silica gel.
The structures of 6 and 7 were assigned based on NMR experiments car-
ried out on the corresponding deblocked products 8 and 9. For NMR stud-
ies, compounds 6 and 7 were heated separately with methanolic ammonia
at 40◦C in steel bomb for 12 h to provide 8 and 9 respectively in good yields.
1
In H NMR, N-CH3 protons chemical shifts for 8 and 9 were appeared as
singlet at 4.12 and 4.00 ppm, respectively. It is noteworthy that the presence
of adjacent C7 carbonyl to the N1-CH3 in compound 8 caused the N1-CH3
protons of 8 to shift 0.12 ppm downfield than the N2-CH3 of 9. On the
other hand, the C3-H protons of 8 and 9 were resonated at 8.08 and 8.25
ppm, respectively. The downfield shift of C3-H proton in 9 is expected,[14–16]
because of the shielding effect induced by the N2-CH3 of 9. Additional evi-
dence for the assignment of structures was furnished by consideration of the
2D ROSEY experiments conducted on 8 and 9. As anticipated, 2D ROSEY
experiments of 8 and 9 in DMSO-d6 with a short mixing time of 200 ms
detected ROE between N2-CH3 and C3-H protons in structure 9 but not for
structure 8 (Figure 2).

Solid-phase Parallel Synthesis of Nucleosides
1951
SCHEME 1 Reagents and conditions: (i) NaH/MeI; (ii) MeOH/NH3/110 C; (iii) Pd/C/MeOH/
NH4OH; (iv) Urea/220 C; (v) HMDS/135 C; (vi) 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose/TMS
triflate; (vii) NaOMe/MeOH/Py.

1952
K. S. Ramasamy et al.
FIGURE 2 ROESY spectra were recorded at 32 C, with mixing time of 200 ms. 1H assignments are
also based on 2D TOCSY and 1H-13C HMQC experiments.
After establishing the structures of 6 and 7, they were transformed to
the key intermediates 12 and 15 as shown in Schemes 2 and 3, respectively.
Treatment of 6 with phosphorus pentasulfide in anhydrous dioxane at re-
flux proceeded smoothly to afford 1-methyl-4-(2,3,5-tri-O-benzoyl- -D-ribo-
furanosyl)-5-oxo-pyrazolo[4,3-d]pyrimidine-7(1H,6H)-thione (10) in 60%
yield. Deprotection of the benzoyl groups of 10 with sodium methoxide in

Solid-phase Parallel Synthesis of Nucleosides
1953
SCHEME 2 Reagents and conditions: (i) P2S5/Dioxane; (ii) NaOMe/MeOH; (iii) MeI/NNDIPEA.
SCHEME 3 Reagents and conditions: (i) Lawesson’s regent; (ii) NaOMe/MeOH; (iii) MeI/NNDIPEA.

1954
K. S. Ramasamy et al.
methanol-pyridine mixture gave 11 as yellow crystals. Transformation of 11
into 12 was accomplished by a selective alkylation of the thiol group of 11
with methyl iodide in the presence of N,N-diisopropylethylamine. Similarly,
compound 15 was prepared from 7 by following the reaction conditions
used for the synthesis of 12 with a minor modification in the transforma-
tion of 13 from 7. Accordingly, heating of 2-methyl-4-(2,3,5-tri-O-benzoyl- -
D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine-5,7(1H,6H)-dione 7 with Lawes-
son’s reagent[17] in anhydrous pyridine provided the corresponding thio
derivative 13 in 80% yield. Deblocking of the benzoyl protecting groups in
13 with sodium methoxide followed by methylation of the corresponding
intermediate 14 provided another scaffold 15 in good yield.
Loading of the Pyrazolopyrimidine Nucleoside Scaffolds
on MMT Resin
The synthesis of resin bound scaffolds B (16) and C (17) is outlined in
Schemes 4 and 5, respectively. Polystrene MMT-Cl resin was stirred with 12 in
SCHEME 4 Reagents and conditions: (i) MMTCl resin/2,6-leutidine/DMF; (ii) 1.5 M amines in NMP/
80 C; (iii) 30% HFIP/50 C.

Solid-phase Parallel Synthesis of Nucleosides
1955
anhydrous DMF in the presence of excess 2,6-lutidine for 3 days under inert
atmosphere. MMT resin was selectively loaded on the 5ꢁ-hydroxyl group of
the scaffold B. The loading efficency (66%) was estimated based on the
increase in weight of the starting resin. Similarly, the nucleoside 15 was also
loaded on MMT resin in 74% yield.
Utilizing solid-phase chemistry the scaffold B was validated by synthe-
sizing ten new library members which were then used as reference con-
ditions during the production of actual library on the synthesizer. Table 1
shows the ten amine building blocks used for validation and the results. Fifty
milligrams of the Scaffold B was placed in ten separate wells of a 48 reac-
tion vessel (LabTech, Advanced ChemTech) and ten different amines were
added. The reaction block was heated at 80◦C for 24 h with gentle agita-
tion. The reaction block was cooled to room temperature and the reagents
were drained. The resin was washed with DMF, MeOH, and CH2Cl2, and
dried. To the dried resin 30% hexafluoro isopropanol (HFIP, 1.5 mL) in
CH2Cl2 was added to each well and heated at 50◦C for 24 h. The reaction
SCHEME 5 Reagents and conditions: (i) MMTCl resin/2,6-leutidine/DMF; (ii) 1.5 M amines in NMP/
80 C; (iii) 30% HFIP/50 C.

1956
K. S. Ramasamy et al.
TABLE 1 Validation of Scaffold B with
Ten Different Amines
LC-MSpurity
of products
(%)
Entry
Amines
a
90
71
b
c
3
81
75
92
d
e
f
g
h
i
36
0
0
j
0
mixture was filtered into ten different vials. The filtrates were evaporated,
and the residues obtained were analyzed by LC-MS. Preliminary validation
results indicated (Table 1) that high quality products were obtained when
primary amines were used. Amines such as secondary amines, hydrazines,
piperazines, and sterically hindered amines did not react well, and they were
eliminated in the final production. Scaffold C behaved differently on vali-
dation with ten different amines (Table 2). N,N-dimethylformamide (DMF)

Solid-phase Parallel Synthesis of Nucleosides
1957
TABLE 2 Validation of Scaffold C with
Ten Different Amines
LC-MSpurity
products
Entry
Amines
of (%)
a
47
b
c
79
100
82
d
e
f
64
8
g
7
h
65
i
j
2
0
was found to be useful for these reactions, but, the formation of side prod-
ucts was noted. Therefore, DMF was not used for library production. After
experimenting with different solvents, 1-methyl-2-pyrrolidinone (NMP) was
found to be suitable for nucleoside library production using solid-phase
chemistry.

1958
K. S. Ramasamy et al.
Based on availability, diversity, lipophilicity, and reactivity of the amine
building blocks, 96 primary amines were selected. Simultaneously, 96 wells
of reactions were carried out for each scaffold (2
96) in one reaction
block on the ACT Vanguard semi-automated synthesizer. The various amines
selected for the final library production are shown in Figure 3. The 96
compounds in different wells were enumerated by the Afferent TeamWorks
software to generate the molecular weight, structural and other related
information for the desired library members, and the key impurities found
FIGURE 3 Building blocks: 96 amines used for the nucleophilic reactions with scaffolds B and C to
provide the corresponding products B1-B96 and C1-C96.

Solid-phase Parallel Synthesis of Nucleosides
1959
in each reaction well. Afferent TeamWorks transferred the data to Label
Automador for automated sample bar code labeling and weighing. Libraries
(Schemes 4 and 5) were synthesized in a parallel fashion utilizing the
optimized procedures and conditions as well as using NMP as solvent. The
washing procedures were automatically conducted and controlled by the
pre-designed program.
Cleavage of Nucleosides from the MMT Resin
A variety of cleavage conditions were explored to obtain the highest
purity of the cleaved nucleoside products. Although 2% TFA in CH2Cl2 had
been utilized to cleave nucleosides from MMT resin,[12] application of this
cleavage condition for pyrazolopyrimidine nucleosides resulted in glycosidic
bond cleavage and therefore reduction in product purity. After exploration
of a range of TFA/CH2Cl2 (0.5–2.0%) mixtures, it was found that 0.5%
TFA in CH2Cl2 was the ideal reagent to cleave the pyrazolopyrimidine
nucleosides from the MMT resin. However, application of the same cleavage
condition (0.5% TFA/CH2Cl2) to the products derived from scaffold C
again resulted in glycosidic bond cleavage of a few nucleosides. Hence, a
different reagent to cleave the MMT resin containing pyrazolopyrimidine
nucleosides was sought. After a few attempts with other reagents (data not
shown), it was found that 30% hexafluoroisopropanol (HFIP) is sufficient
to cleave the nucleosides from the MMT resin and provided good recovery
of the products with acceptable purity. Remarkably, based on the results
obtained the HFIP cleavage condition is the best to use for the acid sensitive
nucleosides.
In all cleavage procedure, the wells containing the resin were treated
with 30% HFIP (1.5 mL) and shaken at 50◦C for 24 h. The mixture was
cooled to room temperature, filtered and washed with MeOH (2 mL), and
the combined filtrate was dried using Savant SpeedVac and the residue
was analyzed by high-throughput LC-MS. The Afferent TeamWorks software
generated compound ID numbers and molecular weights, which were input
into the sample list for LC-MSdata acquisition. The LC-MSdata were
acquired using MassLynx software, and the LC-MSdata were processed
using OpenLynx software. The LC-MSdata and the compound purity
summary were input into Afferent TeamWorks as a record for the database.
Table 3 shows the quality of nucleoside libraries that were produced from 96
amines (Figure 3) with scaffolds B and C using solid-phase parallel synthesis
methodology. LC-MSresults (Table 3) indicate that 78% of library members
(B1-B96)[18] are more than 80% purity and are registered in the data base
directly without purification. The library with more than 80% purity was
considered a good quality library and a successful demonstration of the
use of solid phase chemistry to generate nucleoside libraries. Hence, the

1960
K. S. Ramasamy et al.
TABLE 3 Quality of Libraries (B1-B96 and C1-C96) that Obtained from
Scaffolds B and C on Reaction with 96 Amines
% of products
80–100% purity
% of products
60–80% purity
% of products
>60% purity
Scaffolds
Scaffold B
Scaffold C
78.1
93.7
9.4
4.1
87.5
97.8
96 Compounds for each library.
library with more than 80% purity was selected by the programmed software
and automatically registered to the database for biological screening. 9.4%
of library members (B1-B96)[18] have more than 60% purity and these
members were purified by HPLC and submitted for testing. In total, 88%
of the library production was considered a success from scaffold B. On the
other hand the library (C1-C96)[18] produced from the scaffold C has a
success rate of 98% (Table 3). In both cases, an average of 12 to 20 mg
individual library member was obtained.
In conclusion, we have prepared new pyrazolopyrimidine nucleosides,
assigned their structures by NMR studies, transformed them to the key
intermediates (12 and 15) and loaded on MMT resin efficiently. The
resulting scaffolds B and C were then subjected to nucleophilic substitution
reactions with a variety of amines at the solid-phase level. Subsequently,
the products were cleaved from the solid support under neutral condition,
without cleavage of the glycosidic bond, to provide quality nucleoside
libraries. Thus, the solid-phase parallel synthesis methodology discussed
here is straightforward and opens a new avenue to generate a wide variety of
nucleoside combinatorial libraries. Biological screening of these nucleoside
libraries is in progress and will be reported elsewhere.
EXPERIMENTAL
All 1D NMR spectra were recorded at 300 MHz and the chemical shifts
are referenced to trimethylsilane. All 2D NMR experiments were carried
out on a Bruker Avance DRX 500 system with proton carrier frequency at
500.13 MHz. Data were acquired with 5000 Hz spectral width, 8 transients,
3 seconds delay and 512 data points in t1 dimension. Fourier-transform in-
frared (FT-IR) spectra of the samples on solid support were obtained on a
Perkin-Elmer FT-IR spectrometer. The libraries were enumerated by Affer-
ent TeamWorks 3.0, labeled, and weighed by Label Automador, and synthe-
sized on the ACT Vanguard semi-automated synthesizer. Libraries were ana-
lyzed on a LC-MSsystem. The LC-MSsystem consists of Waters 2790 HPLC,
Waters 996 photodiode array (PDA) detector, and Micromass/Waters ZQ
mass spectrometer. Luna C18 column from Phenomenex was used for com-
pound separation. The mass spectra at m/z 100–1000 were acquired using

Solid-phase Parallel Synthesis of Nucleosides
1961
electrospray ionization with both positive and negative ion detections. UV
spectra were recorded at 200–400 nm by the PDA, and the compound pu-
rity was monitored based on the UV absorbency at 220 nm. The LC-MS
operation was controlled by MassLynx software, and the LC-MSdata were
processed by OpenLynx software. High resolution mass spectra (HRMS)
were acquired using a Q-tof-2 mass spectrometer equipped with an electro-
spray ionization source. Polystyrene monomethoxytrityl chloride resin was
purchased from Novabiochem. Other starting materials, building blocks,
and reagents were purchased from Aldrich and other companies, and used
directly.
Ethyl 4-Nitro-1- and -2-(Methyl)-1H- and 2H-Pyrazole-3-carboxylate (2).
To a stirred solution of ethyl 4-nitro-1H-pyrazole-3-carboxylate (1, 25.00 g,
135.0 mmol) in anhydrous DMF (200 mL) under an argon atmosphere was
added NaH (6.00 g, 150.0 mmol) for 30 min at 0◦C. After the addition of
NaH, the reaction mixture was stirred at room temperature for 1 h and
cooled to 0◦C. Methyl iodide (15 mL) was added slowly and the stirring
continued at 0◦C for 1 h and at room temperature for 6 h. The reaction
mixture was evaporated to dryness. The residue was partitioned between
EtOAc (500 mL) and water (300 mL), and extracted with EtOAc. The
organic layer was washed with brine (150 mL), dried, and concentrated to
dryness. The residue was used as such for the next reaction. Yield: 26.8 g
(100%). UV (MeOH)
273 (5250) and 275.(5100). Anal. calcd. for
max
C7H9N3O4: C, 42.21; H, 4.56; N, 21.09. Found: C, 42.41; H, 4.53; N, 21.25.
4-Nitro-1- and -2-(Methyl)-1H- and 2H-Pyrazole-3-carboxamide (3). A
mixture of compound 2 and methanolic ammonia (prepared freshly at 0◦C,
400 mL) was allowed to stir in a steel bomb at 110◦C for 12 h. The steel
vessel was cooled in dry ice/acetone bath for 1 h, opened carefully, and the
content was evaporated to dryness. The residue was triturated with acetone
and filtered. The solid was used as such for further reaction. Yield: 22.97 g
(100%). 1H NMR (CDCl3): 3.84 and 3.88 (3H, 2 s, NCH3), 7.72–8.8 (3H,
m, CONH2 and ArH). UV (MeOH)
272 (5200) and 278 (5300). Anal.
max
calcd. for C5H6N4O3: C, 35.29; H, 3.55; N, 32.93. Found: C, 35.40; H, 3.73;
N, 32.95.
1- and -2-(Methyl)-1H- and 2H-Pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-
dione (5). To a solution of 3 (22.97 g, 115.4 mmol) in a mixture of
MeOH/dioxane (9:1, 300 mL) was added NH4OH (20 mL) followed by
Pd/C (5%, 1.00 g). The reaction mixture was hydrogenated at 45 psi of
hydrogen for 12 h. The catalyst was filtered, washed with MeOH (100 mL),
and the filtrate evaporated to dryness. The residue 4 that obtained was used
as such for the next reaction without characterization.

1962
K. S. Ramasamy et al.
The above crude product 4 (18.92 g, 135.0 mmol) and urea (25.00 g)
were mixed together and heated at 250◦C with stirring for 45 min. The
product formed as a liquid and then solidified. The solid was dissolved in
2 M NaOH solution (100 mL) at 70◦C and filtered while hot. The filtrate
was acidified with AcOH and cooled. The precipitated solid was filtered,
washed with water and dried over solid NaOH under vacuum. Yield: 22.41 g
1
(100%). H NMR (DMSO-d6): 3.91 and 4.02 (3H, 2 s, NCH3), 7.32 and
7.62 (1H, 2 s, ArH), 10.86 (2H, br s, 2 NH). UV (MeOH) max 285 (5450)
and 290 (5650).
1-Methyl-4-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)pyrazolo[4,3-d]pyrim-
idine-5,7(4H,6H)-dione (6) and 2-Methyl-4-(2,3,5-tri-O-benzoyl-β-D-ribofu-
ranosyl)pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione (7). A mixture of 5
(18.30 g, 110.0 mmol), ammonium sulfate (0.4 g), 1,1,1,3,3,3-hexamethyldi-
silazane (200 mL), and anhydrous pyridine (50 mL) was heated at 135◦C for
12 h under argon and evaporated to dryness. The residue was co-evaporated
with dry toluene (2 100 mL) and dissolved in dry CH3CN (200 mL). 1-O-
Acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose (65.59 g, 130.0 mmol) was added
and cooled to 0◦C under argon. Trimethylsilyl trifluoromethanesulfonate
(TMStriflate, 28.1 mL, 158.4 mmol) was added slowly during 30 min. The
reaction mixture was stirred at 0◦C for 1 h and at room temperature for 12 h.
The solution was evaporated to dryness, partitioned between EtOAc (500
mL) and sat. NaHCO3 (300 mL), and extracted with EtOAc. The organic
extract was washed with water (200 mL) and brine (200 mL), dried and
evaporated to dryness. The residue was purified by flash chromatography
over silica gel using CHCl3
EtOAc as the eluent. Two products (6 and 7)
1
were isolated. Yield of 6: 27.0 g (40%); H NMR (DMSO-d6):ꢁ 4.02 (3H,
ꢁ
ꢁ
ꢁ
s, NCH3), 4.69 (3Hꢁ, m, C5 -CH2 and C4 -H), 6.01 (2H, m, C2 -H and C3 -
H), 6.34 (1H, d, C1 -H), 7.40–8.02 (16H, m, ArH), 11.66 (1H, s, NH). UV
(MeOH)
282 (9200). Anal. calcd. for C32H26N4O9: C, 62.95; H, 4.29;
max
N, 9.18. Found: C, 63.15; H, 4.33; N, 9.09. HRMS(ESI-Q-tof-2) m/z 633.1622
(633.1597 calcd for C32H26N4O9Na [M+Na]+). Yield of 7: 36.93 g (56%);
ꢁ
ꢁ
1H NMR (DMSO-d6)ꢁ: 3.82 (3H, s, NCH3), 4.66 (3H, m, C5 -CH2 and C4 -
ꢁ
ꢁ
H), 5.98 (2H, m, C2 -H and C3 -H), 6.28 (1H, d, C1 -H), 7.30–8.12 (16H,
m, ArH), 11.38 (1H, s, NH). UV (MeOH)
280 (8000). Anal. calcd.
max
for C32H26N4O9: C, 62.95; H, 4.29; N, 9.18. Found: C, 62.71; H, 4.13; N,
9.29. HRMS(ESI-Q-tof-2) m/z 633.1616 (633.1597 calcd for C32H26N4O9Na
[M+Na]+).
1-Methyl-4-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidine-5,7(1H,6H)-
dione (8). Compound 6 (1.00 g, 1.6 mmol) was stirred in methanolic am-
monia (saturated at 0◦C, 70 mL) in a pressure bottle for 12 h at room
temperature. The pressure bottle was cooled to 0◦C, opened, and the

Solid-phase Parallel Synthesis of Nucleosides
1963
content was evaporated to dryness. The residue was triturated with EtOAc
(20 mL) and the EtOAc extract was discarded. The residue on trituration
with MeOH gave colorless powder, which was filtered. Yield: 0.42 g (86%).
1H NMR (500 MHz; DMSO-d6): 3.66 (2H, s), 3.86 (1H, s), 4.12 (3H, s,
ꢁ
NCH3), 4.30 (1H, s), 5.12–5.30 (3H, m, 3 OH), 6.04 (1H, s, C1 -H), 8.08
(1H, s, ArH), 11.54 (1H, s, NH). UV (MeOH)
283 (5200). Anal. calcd.
max
for C11H14N4O6: C, 44.29; H, 4.73; N, 18.78. Found: C, 44.17; H, 4.41; N,
18.90. HRMS(ESI-Q-tof-2) m/z 321.0821 (321.0811 calcd for C11H14N4O6Na
[M+Na]+).
2-Methyl-4-β-D-ribofuranosylpyrazolo[4,3-d]pyrimidine-5,7(1H,6H)-
dione (9). Compound 7 (0.61 g, 1.0 mmol) was stirred in methanolic am-
monia (saturated at 0◦C, 70 mL) in a pressure bottle for 12 h at room
temperature. The pressure bottle was cooled to 0◦C, opened, and the
content was evaporated to dryness. The residue was triturated with EtOAc
(20 mL) and the EtOAc extract was discarded. The residue on trituration
with MeOH gave a colorless powder, which was filtered. Yield: 0.27 g (90%).
1H NMR (500 MHz; DMSO-d6): 3.72 (2H, s), 3.86 (1H, s), 4.00 (3H, s,
NCH3), 4.08 (1H, s), 4.28 (1H, s), 5.08–5.36 (3H, m, 3 OH), 6.02 (1H, s,
ꢁ
C1 -H), 8.25 (1H, s, ArH), 11.30 (1H, s, NH). UV (MeOH) max 278 (4850).
Anal. calcd. for C11H14N4O6: C, 44.29; H, 4.73; N, 18.78. Found: C, 44.07;
H, 4.71; N, 18.59. HRMS(ESI-Q-tof-2) m/z 321.0827 (321.0811 calcd for
C11H14N4O6Na [M+Na]+).
1-Methyl-4-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-oxo-pyrazolo[4,3-d]
pyrimidine-7(1H,6H)-thione (10). A mixture of 6 (10.0 g, 16.4 mmol) and
P2S5 (10.0 g) in anhydrous dioxane (100 ml) was heated at reflux for
12 h under argon and evaporated to dryness. The residue was partitioned
between EtOAc (300 mL) and water (200 mL), and extracted with EtOAc.
The organic extract was washed with brine (200 mL), dried, and evaporated
to dryness. The residue was purified by flash chromatography over silica gel
using CHCl3
EtOAc as the eluent. The pure fraction were pooled and
concentrated to dryness to give the titled compound 10 as yellow foam.
1
Yield:ꢁ7.00 g (68%). H NMR (DMSO-dꢁ6): 4.27 (3H, s, NCH3), 4.71 (3H,
ꢁ
ꢁ
ꢁ
m, C5 -CH2 and C4 -H), 6.04 (2H, m, C2 -H and C3 -H), 6.35 (1H, d, C1 -H),
7.40–8.04 (16H, m, ArH), 12.89 (1H, s, NH). UV (MeOH) max 266 (4250).
Anal. calcd. for C32H26N4O8S: C, 61.34; H, 4.18; N, 8.94; S, 5.11. Found: C,
61.44; H, 4.31; N, 9.00; S, 5.26. HRMS (ESI-Q-tof-2) m/z 649.1354 (649.1369
calcd for C32H26N4O8SNa [M+Na]+).
1-Methyl-4-β-D-ribofuranosyl-5-oxo-pyrazolo[4,3-d]pyrimidine-7(1H,
6H)-thione (11). Compound 10 (10.0 g, 15.9 mmol) was dissolved in
anhydrous pyridine (100 mL) and stirred at room temperature. To this

1964
K. S. Ramasamy et al.
stirred solution was added anhydrous MeOH (100 mL) followed by 25%
wt. NaOMe solution (15 mL) in MeOH. The reaction mixture was stirred
at room temperature for 2 h and neutralized with H+ (Dowex) resin. The
resin was filtered and washed with pyridine (50 mL) and MeOH (50 mL).
The filtrate was concentrated to dryness. The residue was triturated with
MeOH (minimum) and cooled to 0◦C. The precipitated solid was filtered
and washed with minimum amount of MeOH. Yield: 4.22 g (84%). 1H NMR
ꢁ
(DMSO-d6): 3.62 (2H, m, C5 -CH2), 3.80 (1H, m), 4.02 (1H, m), 4.27 (3H,
s, NCH3), 5.02 (1H, m, OH), 5.10 (1H, m, OH), 5.14 (1H, m, OH), 6.00
ꢁ
(1H, m, C1 -H), 8.13 (1H, s, ArH), 12.70 (1H, s, NH). UV (MeOH)
max
258 (4650). Anal. calcd. for C11H14N4O5S: C, 42.04; H, 4.49; N, 17.83; S,
10.18. Found: C, 42.31; H, 4.39; N, 17.66; S, 10.06. HRMS (ESI-Q-tof-2) m/z
337.0594 (337.0583 calcd for C11H14N4O5SNa [M+Na]+).
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-thiomethyl-pyrazolo[4,3-d]pyrimi-
dine (12). To a stirred solution of 11 (5.00 g, 15.9 mmol) in anhydrous DMF
(100 mL) at 0◦C was added N,N-diisopropylethylamine (2.58 g, 20.0 mmol).
To this stirred solution was added MeI (5.0 mL) and the stirring continued
at 0◦C for 1 h and at room temperature for 12 h. The reaction mixture was
evaporated to dryness. The residue was triturated with MeOH (minimum)
and cooled to 0◦C. The precipitated solid was filtered and washed with
1
minimum amount of MeOH. Yield: 4.50 g (86%). H NMR (DMSO-d6):
ꢁ
2.78 (3H, s, SCH3), 3.62 (2H, m, C5 -CH2), 3.80 (1H, m), 4.04 (1H, m),
4.16 (3H, s, NCH3), 4.24 (1H, m), 5.04–5.24 (3H, m, 3 OH), 6.06 (1H, d,
ꢁ
C1 -H), 8.08 (1H, s, ArH). Anal. calcd. for C12H16N4O5S: C, 43.90; H, 4.91;
N, 17.07; S, 9.75. Found: C, 43.91; H, 4.83; N, 17.26; S, 9.76. HRMS (ESI-Q-
tof-2) m/z 351.0746 (351.0739 calcd for C12H16N4O5SNa [M+Na]+).
2-Methyl-4-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-5-oxo-pyrazolo[4,3-d]
pyrimidine-7(1H,6H)-thione (13). A mixture of 7 (18.3 g, 30.0 mmol) and
Lawessons’ reagent (24.24 g, 60.0 mmol) in anhydrous pyridine (300 mL)
was heated at reflux for 12 h under argon and evaporated to dryness. The
residue was suspended in EtOAc (300 mL) and water (200 mL) and treated
with sat. NaHCO3 solution to pH 7. The aqueous layer was extracted with
EtOAc (2
mL), dried, and evaporated to dryness. The residue was purified by flash
chromatography over silica gel using CHCl3 EtOAc as the eluent. The
150 mL). The organic extract was washed with brine (200
pure fraction were pooled and concentrated to dryness to give the titled
1
compound 13 as yellowish foam. Yield: 18.7 ꢁg (99%). Hꢁ NMR (DMSO-
d6): 3.84 (3H,ꢁ s, NCH3), 4.70 (3H, m, C5 -CH2 and C4 -H), 5.98 (2H,
ꢁ
ꢁ
m, C2 -H and C3 -H), 6.32 (1H, d, C1 -H), 7.34–8.12 (16H, m, ArH), 12.68
(1H, s, NH). UV (MeOH)
262 (4050). Anal. calcd. for C32H26N4O8S:
max
C, 61.34; H, 4.18; N, 8.94; S, 5.11. Found: C, 61.11; H, 4.39; N, 9.17; S,

Solid-phase Parallel Synthesis of Nucleosides
1965
5.05. HRMS(ESI-Q-tof-2) m/z 649.1360 (649.1369 calcd for C32H26N4O8SNa
[M+Na]+).
2-Methyl-4-β-D-ribofuranosyl-5-oxo-pyrazolo[4,3-d]pyrimidine-7(1H,
6H)-thione (14). Compound 13 (18.7 g, 29.8 mmol) was dissolved in
anhydrous pyridine (100 mL) and stirred at room temperature. To this
stirred solution was added anhydrous MeOH (100 mL) followed by 25%
wt NaOMe solution (20 mL) in MeOH. The reaction mixture was stirred
at room temperature for 2 h and neutralized with H+ (Dowex) resin. The
resin was filtered and washed with pyridine (50 mL) and MeOH (50 mL).
The filtrate was concentrated to dryness. The residue was triturated MeOH
(minimum) and cooled to 0◦C. The precipitated solid was filtered and
1
washed with minimum amount of MeOH. Yield 8.5 g (91%). H NMR
ꢁ
(DMSO-d6): 3.62 (2H, m, C5 -CH2), 3.82 (1H, m), 3.94 (3H, s, NCH3),
4.00 (1H, m), 4.22 (1H, m),ꢁ 5.07 (1H, m, OH), 5.24 (1H, m, OH), 5.27
(1H, m, OH), 5.98 (1H, d, C1 -H), 8.21 (1H, s, ArH), 12.58 (1H, s, NH). UV
(MeOH)
256 (4350). Anal. calcd. for C11H14N4O5S: C, 42.04; H, 4.49;
max
N, 17.83; S, 10.18. Found: C, 42.07; H, 4.12; N, 17.86; S, 10.23. HRMS (ESI-
Q-tof-2) m/z 337.0591 (337.0583 calcd for C11H14N4O5SNa [M+Na]+).
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-thiomethyl-pyrazolo[4,3-d]pyrimi-
dine (15). To a stirred solution of 14 (8.40 g, 26.7 mmol) in anhydrous DMF
(120 mL) at 0◦C was added N,N-diisopropylethylamine (3.87 g, 30.0 mmol).
To this stirred solution was added MeI (10 mL) and the stirring continued
at 0◦C for 1 h and at room temperature for 12 h. The reaction mixture was
evaporated to dryness. The residue was triturated with MeOH (minimum)
and cooled to 0◦C. The precipitated solid was filtered and washed with
1
minimum amount of MeOH. Yield: 8.20 g (93%). H NMR (DMSO-d6):
ꢁ
2.54 (3H, s, SCH3), 3.64 (2H, m, C5 -CH2), 3.82 (1H, m), 3.98 (3H, s,
NCH3), 4.02 (1H, m), 4.20 (1H, m), 5.06 (1H, d, OH), 5.20 (2H, m, 2
ꢁ
OH), 6.04 (1H, d, C1 -H), 8.20 (1H, s, ArH). Anal. calcd. for C12H16N4O5S:
C, 43.90; H, 4.91; N, 17.07; S, 9.75. Found: C, 43.84; H, 4.97; N, 17.31; S,
9.56. HRMS(ESI-Q-tof-2) m/z 351.0756 (351.0739 calcd for C12H16N4O5SNa
[M+Na]+).
1-Methyl-4-(5) -O-monomethoxytrityl-polystyrene Resin-β-D-ribofurano-
syl)-5-oxo-7-thiomethyl-pyrazolo[4,3-d]pyrimidine (16). Scaffold B. To a
suspension of polystyrene monomethoxytrityl chloride resin (7.5 g, 1.8
mmol/g) in anhydrous DMF (30 mL) was added compound 12 (5.02 g, 15.8
mmol) followed by 2,6-lutidine (3.68 g, 33.6 mmol). The reaction mixture
was shaken well at room temperature for 3 days under an inert atmosphere.
The resin was quenched with MeOH (5 mL) and filtered, and sequentially
washed with anhydrous DMF (3
20 mL), CH2Cl2 (3
20 mL), MeOH

1966
K. S. Ramasamy et al.
(3 20 mL), and CH2Cl2 (3 20 mL). The resin was dried under vacuum
over solid KOH for 12 h. The loading efficiency (66%) was calculated based
on increase in weight of the resin. FT-IR (KBr) 1701 (C=O), 1248 (S-CH3)
cm−1
.
2-Methyl-4-(5) -O-monomethoxytrityl-polystyrene Resin-β-D-ribofurano-
syl)-5-oxo-7-thiomethyl-pyrazolo[4,3-d]pyrimidine (17). Scaffold C. To a
suspension of polystyrene monomethoxytrityl chloride resin (11.11 g, 1.8
mmol/g) in anhydrous DMF (25 mL) was added compound 15 (7.90 g, 24.1
mmol) followed by 2,6-lutidine (12.89 g, 120.5 mmol). The reaction mixture
was shaken well at room temperature for 4 days under an inert atmosphere.
The resin was quenched with MeOH (10 mL) and filtered, and sequentially
washed with anhydrous DMF (3
20 mL), CH2Cl2 (3
20 mL), MeOH
(3 20 mL) and CH2Cl2 (3 20 mL). The resin was dried under vacuum
over solid KOH for 12 h. The loading efficiency (74%) was calculated based
on increase in weight of the resin. FT-IR (KBr) 1700 (C=O), 1249 (S-CH3)
cm−1
.
General Procedure for the Synthesis of
Pyrazolopyrimidine Libraries
Approximately (50 mg) of the resin 16 was dispensed in each of the 96
reaction wells using a spatula and funnel. The respective amines (1.5 M in
NMP, 1.5 mL) were added to the corresponding wells. The reaction flask
was covered and placed in the synthesizer. The reaction conditions were
programmed and turned on. The reaction block was heated at 80◦C with
shaking for 24 h. The reaction block was cooled to room temperature and
the solvent was drained. The resin was washed with DMF (3 1 mL), MeOH
(3 1 mL) and CH2Cl2 (3 1 mL), and dried under nitrogen.
Cleavage of the Nucleosides from Resin
A 30% solution of HFIP in CH2Cl2 (1.5 mL) was added to the resin
in each well. The reaction block was closed and shaken with heating at
50◦C for 24 h. The 96 reaction wells were filtered into 96 pre-labeled and
pre-weighed vials in 96 well format. The 96 wells were washed with MeOH
(1.00 mL) and filtered into the corresponding 96 wells. The filtrates were
concentrated under vacuum using Savant SpeedVac to dryness to provide
12–20 mg of products.
1H NMR and mass spectral data of the selected library members are
given below:
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(2,2-dimethoxy)ethylamino)]-py-
razolo[4,3-d]pyrimidine (B1). 1H NMR (CD3OD): 3.40–3.60 (5H, m),

Solid-phase Parallel Synthesis of Nucleosides
1967
3.64–3.84 (6H, m), 3.92–4.18 (5H, m), 4.22 (1H, m), 4.52 (1H, m), 6.07
(1H, d, J 6.6, C1 -H), 7.93 (1H, m, ArH); MS(ESI) m/z 386 (M+H)+.
ꢁ
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(2-methoxy)ethylamino)]-pyrazo-
lo[4,3-d]pyrimidine (B8). 1H NMR (CD3OD): 3.30 (2H, m), 3.38 (3H, s),
3.65 (2H, m), 3.81 (2H,ꢁm), 3.98 (1H, m), 4.18 (3H, s), 4.24 (1H, m), 4.58
(1H, m), 6.06 (1H, d, C1 -H), 7.90 (1H, s, ArH); MS(ESI) m/z 356 (M+H)+.
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[2-(1-methylpyrrolidin-2-yl)ethyl-
amino)]-Pyrazolo[4,3-d]pyrimidine (B13). 1H NMR (CD3OD): 1.70–2.20
(4H, m), 2.60 (3H, s), 2.78 (1H, m), 3.30 (2H, m), 3.42 (1H, m), 3.62 (2H,
m), 3.82 (2H, m), 4.00 (1H, m), 4.18 (3H, s), 4.24 (1H, m), 4.56 (1H, m),
6.07 (1H, d, C1 -H), 7.92 (1H, s, ArH); MS(ESI) m/z 409 (M+H)+.
ꢁ
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-(cyclopropylamino)-pyrazolo[4,3-
d]pyrimidine (B17). 1H NMR (CD3OD): 0.74 (2H, m), 0.86 (2H, m),
3.78 (2H, m), 3.98 (1H,ꢁm), 4.04 (1H, m), 4.14 (3H, s), 4.24 (1H, m), 4.58
(1H, m), 6.40 (1H, d, C1 -H), 7.92 (1H, s, ArH); MS(ESI) m/z 338 (M+H)+.
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(pyridine-4-yl)methylamino]-py-
razolo[4,3-d]pyrimidine (B32). 1H NMR (CD3OD): 3.80ꢁ(4H, m), 3.98
(1H, m), 4.22 (4H, m), 4.56 (1H, m), 6.06 (1H, d, J 6.6, C1 -H), 7.50 (1H,
m, ArH), 7.92 (1H, s, ArH), 8.08 (1H, d, ArH), 8.46 (1H, d, ArH), 8.69 (1H,
s, ArH); MS(ESI) m/z 389 (M+H)+.
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[2-(N,N-diethylamino)ethylamino]-
pyrazolo[4,3-d]pyrimidine (B43). 1H NMR (CD3OD): 1.10 (6H, m), 2.72
(4H, m), 2.83 (2H, m), 3.60–3.80 (4H, m), 3.98 (1H, m), 4.18 (3H, s), 4.24
ꢁ
(1H, m), 4.56 (1H, m), 6.07 (1H, d, J 6.6, C1 -H), 7.90 (1H, s, ArH); MS
(ESI) m/z 397 (M+H)+.
1-Methyl-4 -β-D-ribofuranosyl-5-oxo-7-[3-(N,N-dimethylamino)propyl-
amino]-pyrazolo[4,3-d]pyrimidine (B55). 1H NMR (CD3OD): 1.95 (2H,
m), 2.46 (6H, s), 2.69 (2H, m), 3.65 (2H, m), 3.80 (2H, m), 3.99ꢁ(1H, m),
4.18 (3H, s), 4.24 (1H, m), 4.56 (1H, m), 6.07 (1H, d, J 6.6, C1 -H), 7.92
(1H, s, ArH); MS(ESI) m/z 383 (M+H)+.
1-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[3-(hydroxy)propylamino]-pyrazo-
lo[4,3-d]pyrimidine (B93). 1H NMR (CD3OD): 1.85 (2H, m), 3.05 (2H,
m), 3.68 (4H, m), 3.98 (1H, m), 4.18 (3H, s), 4.25 (1H, m), 4.54 (1H, m),
6.06 (1H, d, C1 -H), 7.92 (1 H, s, ArH); MS(ESI) m/z 356 (M+H)+.
ꢁ

1968
K. S. Ramasamy et al.
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(2-hydroxy)ethylamino]-pyrazolo
[4,3-d]pyrimidine (C5). 1H NMR (CD3OD): 3.64 (2H, m), 3.76 (2H, m),
3.82 (2H, m), 3.98 (4H, m), 4.22 (1H, m), 4.46 (1H, m), 6.07 (1H, d, J 6.9,
C1 -H), 8.06 (1H, s, ArH); MS(ESI) m/z 342 (M+H)+.
ꢁ
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(2,3-dihydroxy)propylamino]-py-
razolo[4,3-d]pyrimidine (C7). 1H NMR (CD3OD): 3.50–3.80 (4H, m),
3.82 (3H, m), 3.99 (4H, m), 4.22 (1H, m), 4.45 (1H, m), 6.09 (1H, d, J 6.6,
C1 -H), 8.10 (1H, s, ArH); MS(ESI) m/z 372.45 [372 (M+H)+.
ꢁ
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-(cyclopropylamino)-pyrazolo[4,3-
d]pyrimidine (C17). 1H NMR (CD3OD): 0.68 (2H, m), 0.86 (2H, m),
3.04 (1H, m), 3.82 (2H, m), 3.98 (4H, m), 4.22 (1H, m), 4.46 (1H, m), 6.09
(1H, d, J 6.6, C1 -H), 8.06 (1H, s, ArH); MS(ESI) m/z 338 (M+H)+.
ꢁ
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[3-(imidazol-1-yl)propylamino]-py-
razolo[4,3-d]pyrimidine (C26). 1H NMR (CD3OD): 2.18 (2H, m), 3.58
(2H, m), 3.82 (2H, m), 3.98 (1H,ꢁm), 4.01 (3H, s), 4.14 (2H, m), 4.23 (1H,
m), 4.44 (1H, m), 6.09 (1H, d, C1 -H), 6.98 (1H, s, ImH), 7.22 (1H, s, ImH),
7.80 (1H, s, ImH), 8.08 (1H, s, ArH); MS(ESI) m/z 406 (M+H)+.
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(3-methyl)benzylamino)-pyrazolo
[4,3-d]pyrimidine (C37). 1H NMR (CD3OD): 2.31 (3H, s), 3.81 (2H, m),
3.97 (1H, m), 4.01 (3H, s), 4.22 (1H, m), 4.46 (1H, m), 4.72 (2H, m), 6.09
ꢁ
(1H, d, J 6.6, C1 -H), 7.00–7.22 (4H, m, PhH), 8.08 (1H, s, ArH); MS(ESI)
m/z 402 (M+H)+.
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[2-(N,N-diethylamino)ethylamino]-
pyrazolo[4,3-d]pyrimidine (C43). 1H NMR (CD3OD): 1.52 (6H, m), 2.74–
3.00 (6H, m), 3.72 (2H, m), 3.82 (2H,ꢁm), 3.99 (1H, m), 4.01 (3H, s), 4.22
(1H, m), 4.44 (1H, m), 6.08 (1H, d, C1 -H), 8.08 (1H, s, ArH); MS(ESI) m/z
397 (M+H)+.
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(furan-2-yl)methylamino]-pyrazo-
lo[4,3-d]pyrimidine (C45). 1H NMR (CD3OD): 3.82 (2H, m), 3.98 (1H,
m), 4.01 ꢁ(3H, s), 4.24 (1H, m), 4.44 (1H, m), 4.74 (2H, m), 6.11 (1H, d,
J 6.6, C1 -H), 6.35 (2H, s, ArH), 7.42 (1H, s, ArH), 8.09 (1H, s, ArH); MS
(ESI) m/z 378 (M+H)+.
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(pyridine-4-yl)methylamino]-py-
razolo[4,3-d]pyrimidine (C47). 1H NMR (CD3OD): 3.82 (2H, m), 4.02
ꢁ
(4H, m), 4.46 (1H, m), 6.08 (1H, d, J 6.6, C1 -H), 7.53 (2H, br s, PyH), 8.12
(1H, s, ArH), 8.36–8.80 (2H, m, PyH); MS(ESI) m/z 389 (M+H)+.

Solid-phase Parallel Synthesis of Nucleosides
1969
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(2-hydroxy)propylamino]-pyrazo-
lo[4,3-d]pyrimidine (C53). 1H NMR (CD3OD): 1.20 (3H, m), 3.44 (2H,
m), 3.58 (1H, m), 3.82 (2H, m), 3.96–4.04 (3H, m), 4.23 (1H, m), 4.45 (1H,
m), 6.08 (1H, d, C1 -H), 8.10 (1H, s, ArH); MS(ESI) m/z 356 (M+H)+.
ꢁ
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(4-methyl)benzylamino]-pyrazolo
[4,3-d]pyrimidine (C59). 1H NMR (CD3OD): 2.29 (3H, s), 3.82 (2H, m),
3.99 (4H, m), 4.22 (1H, m), 4.48 (1H, m), 4.70 (2H, m), 6.09 (1H, d, J 6.6,
ꢁ
C1 -H), 7.10 (2H, d, PhH), 7.25 (2H, d, PhH), 8.06 (1H, s, ArH); MS(ESI)
m/z 402 (M+H)+.
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(4-hydroxy)butylamino]-pyrazolo
[4,3-d]pyrimidine (C92). 1H NMR (CD3OD): 1.62 (2H, m), 1.73 (2H,
m), 3.60 (4H, m), 3.82 (2H, m), 3.98 (4H, m), 4.22 (1H, m), 4.45 (1H, m),
6.07 (1H, d, J 6.6, C1 -H), 8.06 (1H, s, ArH); MS(ESI) m/z 370 (M+H)+.
ꢁ
2-Methyl-4-β-D-ribofuranosyl-5-oxo-7-[(3-hydroxy)propylamino]-pyrazo-
lo[4,3-d]pyrimidine (C93). 1H NMR (CD3OD): 1.84 (2H, m), 3.63 (4H,
m), 3.82 (2H, m), 3.98 (1H, m), 4.00 (3H, s), 4.22 (1H, m), 4.45 (1H, m),
ꢁ
4.61 (2H, m), 6.06 (1H, d, C1 -H), 8.06 (1H, s, ArH); MS(ESI) m/z 356
(M+H)+.
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18. B1-B96 are the corresponding products obtained by the reaction 96 amines (Figure 3) with
scaffold B. C1-C96 are the corresponding products obtained by the reaction 96 amines (Figure 3)
with scaffold C. 1-96 represents the corresponding amines from Figure 3.