Synthesis of 2′-substituted inosine analogs via unusual masking of the 6-hydroxyl group

Article abstract of DOI:10.1080/15257770.2011.650254

(Chemical Equation Presented) 2′-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylam

Full text of DOI:10.1080/15257770.2011.650254

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Synthesis of 2′-Substituted Inosine
Analogs via Unusual Masking of the 6-
Hydroxyl Group
Fabio Casu a , Rebecca K. Harston a , Maria A. Chiacchio b
Giuseppe Gumina c
&
a Department of Pharmaceutical Sciences, Medical University of
South Carolina, Charleston, South Carolina, USA
b Dipartimento di Scienze Chimiche, Università di Catania, viale A.
Doria 6, Catania, Italy
c Department of Pharmaceutical Sciences, South University School of
Pharmacy, Savannah, Georgia, USA
Version of record first published: 22 Feb 2012
To cite this article: Fabio Casu, Rebecca K. Harston, Maria A. Chiacchio & Giuseppe Gumina (2012):
Synthesis of 2′-Substituted Inosine Analogs via Unusual Masking of the 6-Hydroxyl Group, Nucleosides,
Nucleotides and Nucleic Acids, 31:3, 224-235
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Nucleosides, Nucleotides and Nucleic Acids, 31:224–235, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.650254
SYNTHESIS OF 2ꢀ-SUBSTITUTED INOSINE ANALOGS
VIA UNUSUAL MASKING OF THE 6-HYDROXYL GROUP
Fabio Casu,1 Rebecca K. Harston,1 Maria A. Chiacchio,2
and Giuseppe Gumina3
1Department of Pharmaceutical Sciences, Medical University of South Carolina,
Charleston, South Carolina, USA
2Dipartimento di Scienze Chimiche, Universita` di Catania, viale A. Doria 6,
Catania, Italy
3Department of Pharmaceutical Sciences, South University School of Pharmacy,
Savannah, Georgia, USA
Graphical Abstract:
H3C
CH3
N
N
N
O
N
N
N
N
NH
RO
N
HO
O
O
O
O
Cl
N
RO
O
HO
O
OCH3
OH
N
N
N
1
HO
O
OBn
N
HO
OH
O
N
N
N
NH
O
6-Chloropurine
riboside
R'O
N
N
N
HO
O
O
R'O
OH
HO
O
OH
O
2
Received 28 November 2011; accepted 13 December 2011.
This work supported by South University School of Pharmacy and Medical University of South
Carolina Institutional Research Funds.
Address correspondence to Giuseppe Gumina, Department of Pharmaceutical Sciences, South
University School of Pharmacy, 709 Mall Boulevard, Savannah, GA 31406. E-mail: ggumina@
southuniversity.edu
224

Synthesis of 2 ꢀ-Substituted Inosine Analogs
225
2 ꢁ-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-
2
dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate
was obtained via an unusually facile dimethylamine transfer from dimethylformamide.
Keywords Modified nucleosides; base modification; anticancer nucleosides
INTRODUCTION
The continued success of nucleoside analogs as therapeutic agents in the
past 40 years has driven the development of novel synthetic methodologies
to access highly modified structures.[1] Research in the field of nucleoside-
based compounds has focused for several years on 2ꢁ-deoxy, 2ꢁ,3ꢁ-dideoxy,
arabino analogs and their carbocyclic or heterosubstituted analogs.[2] This
focus was due to the nature of the biological targets, i.e., human or viral DNA
polymerases.[2] However, more recent research has shifted its focus toward
ribonucleosides due to the need to treat new or re-emerging infectious
diseases caused by RNA viruses, among which hepatitis C[3] is today the most
clinically relevant.
The synthesis of modified ribonucleosides is usually achieved via a con-
vergent approach, in which a suitably activated ribose analog is coupled
to a silylated base in Vorbru¨ggen fashion.[4] Derivatives where the ribose
moiety is modified are often prepared from carbohydrates like xylose or ara-
binose rather than ribose.[1c] As part of our studies toward the synthesis of
2ꢁ-modified inosines as potential inhibitors of purine nucleoside phosphory-
lase, we explored the synthesis of 2ꢁ-inosine ether 1 and ester 2[5] (Figure 1)
via a linear approach, in which functionalization of the ribose moiety is
achieved on the nucleoside. When such an approach is attainable, it has the
advantage of providing target molecule in a relatively small number of steps.
2ꢁ-functionalization of nucleosides is also of great interest due to the activity
of 2ꢁ-modified derivatives against hepatitis C.[6]
O
N
O
N
N
N
N
N
NH
NH
O
HO
HO
O
O
O
HO
O
HO
O
OH
OH
O
1
2
FIGURE 1 2ꢁ-Inosine ether 1, and ester 2.

226
F. Casu et al.
RESULTS AND DISCUSSION
Synthesis
Bis-protection of the 3ꢁ,5-diol functionality of ribonucleosides has
been the subject of extensive literature.[7] The methylene-1,3-bis-diisopro
pylsilanyl protecting group in 3 (Scheme 1) has been reported to be sta-
ble under the strongly basic conditions necessary for the alkylation of
the 2ꢁ-hydroxyl group.[8] The protected 6-chloropurine 3 was subjected
to alkylating conditions via conversion to the sodium salt using sodium
bis(trimethylsilyl)amide at −20◦C, and then at room temperature. Surpris-
ingly, the alkylated product was the dimethylaminopurine analog 4, evidently
resulting from the transfer of a dimethylamino group from the solvent N ,N -
dimethylformamide. When commonly used 3ꢁ,5ꢁ-protecting groups, such
as tetraisopropyldisiloxanyl,[1c,9] tert-butyldimethylsilyl,[10] or acetyl[11] were
tried, the reactions did not yield the desired products. Instead, complex mix-
tures of products of partial desilylation or acetyl migration were detected by
TLC analysis and characterized only partially.
Both the alkylation of the 2ꢁ-OH and the substitution of the 6-chlorine
proceeded very smoothly at room temperature. However, the substitution at
the 2ꢁ-position was very rapid, and quenching the reaction after one hour at
−20◦C resulted in the isolation of the 6-chloropurine derivative 5 in good
yield. No product 4 was formed at this temperature. Monitoring the reaction
by TLC was complicated by the fact that the intermediate chloride 5 had the
same Rf as the starting material 3. However, the use of ethyl bromoacetate
SCHEME 1 Synthesis of 2ꢁ-O-carboxymethylinosine 1.

Synthesis of 2 ꢁ-Substituted Inosine Analogs
227
in place of methyl ester allowed the detection of starting material, interme-
diate, and product 6, which was isolated in almost quantitative yield. When
the reaction was started at −20◦C, then warmed up to 0◦C instead of room
temperature, partial conversion to 4 or 6 was observed. The dimethylamine
functionality in 6 was oxidized to give the protected inosine 7. Deprotec-
tion of the silyl diether with tetrabutylammonium fluoride occurred with
concomitant hydrolysis of the ester functionality to give the final product 1,
which was isolated as the tetrabutylammonium salt.
The amination of 3 at low temperature is interesting, because the
dimethylamine transfer from dimethylformamide to a chlorinated hetero-
cycle had been observed before, but only at high temperatures (80–150◦C)
and in the presence of weaker bases, such as triethylamine and potassium
carbonate.[12] One possible mechanism could be the formation of dimethy-
lamine from dimethylformamide via α-elimination catalyzed by very strong
base, although the low temperatures observed in our case would be unusual.
The possibility that dimethylformamide contained significant amounts of
dimethylamine as normal storage decomposition products was ruled out,
because the same solvent was used in the synthesis of 3 and no amina-
tion of the starting material was observed. The use of tetrabutylammonium
iodide may very well facilitate the reaction by in situ conversion of the 6-
chloropurine to 6-iodopurine reactive intermediate. Indeed, nucleophilic
aromatic substitution of 6-chloropurine with iodide at low temperatures has
been described, although in different conditions.[13] All these possibilities
deserve more thorough investigations, which are beyond the scope of the
work presented herein.
For the synthesis of 2, the masked hypoxanthine heterocyclic moiety was
obtained by benzylation of 6-chloropurine to benzyl aryl ether 8 (Scheme 2).
SCHEME 2 Synthesis of 2ꢁ-O-succinylinosine 2.

228
F. Casu et al.
Benzyl protection allows circumventing the basic hydrolytic step necessary to
convert 6-chloropurine to hypoxanthine. Because in this case no strong bases
are required for the acylation of the 2ꢁ-oxygen, the tetraisopropyldisiloxane-
1,3-diyl protecting group could be used for the protection of the 3ꢁ,5ꢁ-diol,
employing the commercially available dichloride. Acylation of alcohol 9
was accomplished by treatment with succinic anhydride. Deprotection of
the resulting 3ꢁ,5ꢁ-bis silyl ether 10 was effectively accomplished in mild
conditions using triethylamine trihydrofluoride, whereas the classic reagent
tetrabutylammonium fluoride proved to be too basic for the ester function
in 10, which was cleaved to give fully deprotected 8. Hydrogenation of the
benzyl group in 10 afforded the desired product 2.
Although the 6-chloropurine/6-benzyloxypurine/hypoxanthine con-
version seems a logical procedure, we could not find this sequence in the
literature.
Neither 1 nor 2 inhibited E. coli or human purine nucleoside phospho-
rylase in concentrations of up to 200 µM.
SUMMARY AND CONCLUSION
We have described two novel routes toward the synthesis of 2ꢁ-
functionalized inosine ethers or esters. These routes feature alternative
protection, deprotection, and reactivity compared to the known literature
methods and allow the synthesis of inosine analogs bypassing aqueous ba-
sic hydrolytic conditions. Therefore, the described methods are compatible
with the presence of the ester functionality. During the functionalization of
the 2ꢁ-position, we observed an unusually mild dimethylamine transfer from
dimethylformamide to 6-chloropurine. This reaction may represent a novel
mild amination and its general applicability will be tested in future studies.
EXPERIMENTAL
General
All reactions were carried out under a positive pressure of argon and
monitored by TLC on uniplates (silica gel) purchased from Analtech Co.
All the reagents, purine nucleoside phosphorylase, and anhydrous solvents
were purchased from commercial sources and used without further purifi-
cation except where noted. Chromatographic purifications were performed
on flash silica gel (particle size 40–63 µm) purchased from Silicycle or TLC-
grade silica gel (particle size 5–15 µm) purchased from Sorbent Technolo-
gies. All solvents for chromatographic purifications were of HPLC grade.
Melting points were determined on a Barnstead Mel-Temp and are uncor-
rected. 1H NMR spectra were recorded on Varian 500 with Me4Si as an inter-
nal standard. Signals are represented as s (singlet), d (doublet), t (triplet),
m (multiplet), or combinations of the all. UV spectra were obtained on

Synthesis of 2 ꢁ-Substituted Inosine Analogs
229
a BECKMAN DU-650 spectrophotometer. Optical rotations were measured
on a Rudolph Research Analytical Autopol IV digital polarimeter. Elemental
analyses were performed by Atlantic Microlabs Inc. (Norcross, GA, USA).
Experimental Procedures
3ꢁ,5ꢁ-O-(Methylene-bis-diisopropylsilane-1,3-diyl)-6-chloropurine
ribo-
side (3). 6-Chloropurine riboside (1.0 g, 3.49 mmol) and imidazole (1.17
g, 17.1 mmol) were dried by co-evaporation with anhydrous pyridine (3 ×
30 mL) and then dissolved in anhydrous dimethylformamide (30 mL). The
mixture was cooled in ice bath and 1,3 dichloro-bis(diisopropylsilyl)methane
(1.27 mL, 4.19 mmol) was added dropwise. The temperature was gradually
increased to rt and the mixture was stirred for 6 h. The reaction mixture was
poured into water (100 mL) and the resulting suspension was extracted with
dichloromethane (3 × 40 mL). The combined organic extracts were washed
with brine (1 × 20 mL), dried over magnesium sulfate, and concentrated
under reduced pressure. The resulting crude was purified by TLC-grade
silica gel flash chromatography (1:6 ethyl acetate/hexanes) to give 3 as col-
orless crystals (1.15 g, yield: 63%). Rf 0.5 (1:9 methanol/dichloromethane);
25
mp 83–85◦C; [α]D −22.29 (c 0.48, CHCl3); UV (λmax = 264 nm, CH3OH);
1H NMR (400 MHz, CDCl3): δ 8.73 (s, 1H), 8.32 (s, 1H), 6.08 (s, 1H,
anomeric), 4.82 (dd, J = 7.9, 5.4 Hz, 1H), 4.54 (d, J = 5.3 Hz, 1H) 4.14–4.06
(m, 2H), 3.95 (dd, J = 12.1, 2.7 Hz), 3.15 (s, 1H, D2O exchangeable),
1.11–1.03 (m, 28H), 0.05 (dd, J = 21.4, 14.2 Hz, 2H); 13C NMR (400 MHz,
CDCl3) δ 151.7, 151.0, 150.5, 144.0, 132.3, 90.0, 81.7, 74.7, 70.3, 61.1, 17.7,
17.6, 17.6, 17.5, 17.5, 17.4, 17.4, 17.3, 17.3, 14.2, 13.9, 13.6, −9.46; Mass
[MH]+ 527; Anal. Calcd for C23H39ClN4O4Si2: C, 52.40; H, 7.46; N, 10.63.
Found: C, 52.54; H, 7.55; N, 10.39.
3ꢁ,5ꢁ-O-(Methylene-bis-diisopropylsilane-1,3-diyl)-2ꢁ-O-(methoxycarbonyl
methyl)-N,N-dimethyladenosine (4). Sodium bis(trimethylsilyl)amide (1
M solution in tetrahydrofuran, 1.14 mL, 1.14 mmol) was added to a
solution of 3 (0.20 g, 0.38 mmol), methyl bromoacetate (0.11 mL, 1.22
mmol), and tetrabutylammonium iodide (0.04 g, 0.11 mmol) in anhydrous
dimethylformamide (8 mL) at −20◦C, and the resulting mixture was stirred
for 4 h at the same temperature. The temperature was then allowed to warm
up gradually to rt and the mixture was stirred at rt for 72 h. The reaction was
quenched with methanol and evaporated under reduced pressure and the
residue poured into 150 mL of water, extracted with dichloromethane (3 ×
20 mL) and washed with brine (2 × 10 mL). The combined organic extracts
were dried over magnesium sulfate, filtered, and concentrated under
reduced pressure. The resulting crude was purified by TLC-grade silica gel
flash chromatography (1:4 to 2:7 ethyl acetate/hexanes) to give 4 as a yellow
solid (0.200 g, yield: 88%). Rf 0.38 (1:9 methanol/dichloromethane); mp
23
75–77◦C; [α]D −57.58 (c 0.37, CHCl3); UV (λmax = 272.5 nm, CH3OH);

230
F. Casu et al.
1H NMR (400 MHz, CDCl3): δ 8.28 (s, 1H), 8.00 (s, 1H), 6.09 (s, 1H,
anomeric), 4.75 (dd, J = 9.4, 4.5 Hz, 1H), 4.63 (d, J = 16.8 Hz, 1H), 4.44
(d, J = 16.6 Hz, 1H), 4.39 (d, J = 4.5 Hz, 1H), 4.21–4.11 (m, 2H), 3.85 (dd,
J = 13.0, 2.5 Hz, 1H), 3.72 (s, 3H), 3.52 (br s, 6H), 1.11–1.00 (m, 28H),
0.01 (d, J = 14.0 Hz, 1H), −0.08 (d, J = 14.2 Hz, 1H); 13C NMR (400 MHz,
CDCl3) δ 170.7, 155.0, 152.4, 149.7, 137.2, 120.9, 88.7, 82.5, 81.1, 70.6, 68.3,
60.6, 52.0, 38.5, 18.1, 17.9, 17.9, 17.8, 17.8, 17.8, 17.7, 14.5, 14.4, 14.2, −9.59;
Mass [MH]+ 608; Anal. Calcd for C28H49N5O6Si2·0.1 EtOAc: C, 55.31; H,
8.14; N, 11.36. Found: C, 55.64; H, 8.16; N, 11.06.
3ꢁ,5ꢁ-O-(methylene-bis-diisopropylsilane-1,3-diyl)-2ꢁ-O-(methoxycarbonyl
methyl)-6-chloropurine riboside (5). Sodium bis(trimethylsilyl)amide
(1 M solution in tetrahydrofuran, 1.88 mL, 1.88 mmol) was added to a
solution of 3 (0.300 g, 0.569 mmol), methyl bromoacetate (0.17 mL, 1.82
mmol) and tetrabutylammonium iodide (0.06 g, 0.17 mmol) in anhydrous
dimethylformamide (30 mL) at −20◦C, and the resulting mixture was
stirred for 1 h at the same temperature. The reaction was quenched with
methanol, gradually warmed up to rt and concentrated in vacuo to a residue
to which 200 mL of water were added and the resulting suspension was
extracted with dichloromethane (4 × 50 mL). The combined organic
extracts were washed with water (1 × 50 mL) and brine (1 × 50 mL),
dried over magnesium sulfate, filtered, and concentrated under reduced
pressure. The resulting crude was purified by TLC-grade silica gel flash
chromatography (13 to 15% ethyl acetate/hexanes) to give 5 as a colorless
25
syrup (0.230 g, yield: 68%). Rf 0.35 (3:7 ethyl acetate/hexanes); [α]D
1
−38.11 (c 1.00, CHCl3); UV (λmax = 264.0 nm, CH3OH); H NMR (400
MHz, CDCl3): δ 8.73 (s, 1H), 8.46 (s, 1H), 6.22 (s, 1H, anomeric), 4.68 (dd,
J = 9.5, 4.5 Hz, 1H), 4.62 (d, J = 16.6 Hz, 1H), 4.46 (d, J = 16.8 Hz, 1H), 4.39
(d, J = 4.5 Hz, 1H), 4.26–4.15 (m, 2H), 3.87 (dd, J = 13.2, 2.5 Hz, 1H), 3.74
(s, 3H), 1.11–1.00 (m, 28H), 0.03 (d, J = 14.0 Hz, 1H), −0.07 (d, J = 14.2
Hz, 1H); 13C NMR (400 MHz, CDCl3): δ 170.3, 151.8, 150.9, 150.5, 144.0,
132.4, 88.9, 82.1, 81.2, 70.1, 68.1, 60.0, 51.8, 17.8, 17.6, 17.6, 17.5, 17.5, 17.4,
14.2, 14.1, 14.0, -9.85; Mass [MH]+ 599; Anal. Calcd for C26H43ClN4O6Si2:
C, 52.11; H, 7.23; N, 9.35. Found: C, 52.32; H, 7.28; N, 9.16.
3ꢁ,5ꢁ-O-(Methylene-bis-diisopropylsilane-1,3-diyl)-2ꢁ-O-(ethoxycarbonyl
methyl)-N,N-dimethyladenosine (6). Sodium bis(trimethylsilyl)amide (1 M
solution in tetrahydrofuran, 4.50 mL, 4.50 mmol) was added to a solution
of 3 (0.790 g, 1.50 mmol), ethyl bromoacetate (0.53 mL, 4.79 mmol),
and tetrabutylammonium iodide (0.170 g, 0.449 mmol) in anhydrous
dimethylformamide (40 mL) at −20◦C. The mixture was stirred for 1 h
at the same temperature, then allowed to warm up gradually to rt and
stirred for 72 h. The reaction was quenched with methanol and evaporated
under reduced pressure and the residue was poured into 200 mL of water,
extracted with dichloromethane (3 × 70 mL), and washed with brine (1 ×
50 mL). The combined organic extracts were dried over magnesium sulfate,

Synthesis of 2 ꢁ-Substituted Inosine Analogs
231
filtered, and concentrated under reduced pressure. The resulting crude
was purified by TLC-grade silica gel flash chromatography (14 to 20% ethyl
acetate/hexanes) to give 6 as a yellow syrup (0.90 g, yield: 97%). Rf 0.6 (1:9
25
methanol/dichloromethane); [α]D −42.60 (c 0.46, CHCl3); UV (λmax
=
1
277.5 nm, CH3OH); H NMR (400 MHz, CDCl3): δ 8.27 (s, 1H), 7.99 (s,
1H), 6.09 (s, 1H, anomeric), 4.77 (dd, J = 9.4, 4.5 Hz, 1H), 4.60 (d, J =
16.6 Hz, 1H), 4.43 (d, J = 16.8 Hz, 1H), 4.41 (d, J = 4.5 Hz, 1H), 4.21–4.10
(m, 4H), 3.84 (dd, J = 13.1, 2.5 Hz, 1H), 3.52 (br s, 6H), 1.23 (t, J = 7.0
Hz, 3H), 1.11–1.00 (m, 28H), 0.02 (d, J = 14.0 Hz, 1H), −0.08 (d, J = 14.2
Hz, 1H); 13C NMR (400MHz, CDCl3) δ 170.1, 154.7, 152.2, 149.4, 137.0,
120.7, 88.5, 82.0, 80.8, 70.3, 68.1, 60.8, 60.3, 38.4, 17.9, 17.7, 17.7, 17.6, 17.6,
17.6, 17.5, 14.2, 14.1, 14.1, 13.9, −9.90; Mass [MH]+ 622; Anal. Calcd for
C29H51N5O6Si2·0.05 Hex: C, 56.20; H, 8.32; N, 11.18. Found: C, 56.55; H,
8.30; N, 10.95.
3ꢁ,5ꢁ-O-(Methylene-bis-diisopropylsilane-1,3-diyl)-2ꢁ-O-(ethoxycarbonyl
methyl)inosine (7). A mixture of 6 (0.160 g, 0.257 mmol) and MCPBA
(0.288 g, 1.29 mmol) in chloroform (10 mL) was stirred at 50◦C for
24 h. The mixture was concentrated under reduced pressure to a
crude that was purified by TLC-grade silica gel flash chromatography
(dichloromethane to 1:19 methanol/dichloromethane), then recrystallized
from ethanol to afford pure 7 as a white solid (0.100 g, 65%): Rf 0.17
27
(1:19 methanol/dichloromethane); mp 136–138◦C; [α]D −37.95 (c 0.50,
CHCl3); UV (λmax = 245 nm, 269.5 nm (sh), CH3OH); 1H NMR (400 MHz,
CDCl3): δ 13.13 (br s, 1H, D2O exchangeable), 8.18 (s, 1H), 8.08 (s, 1H),
6.15 (s, 1H, anomeric), 4.62 (dd, J = 9.4, 4.4 Hz, 1H), 4.57 (d, J = 16.8 Hz,
1H), 4.45 (d, J = 16.6 Hz, 1H), 4.29 (d, J = 4.3 Hz, 1H), 4.25–4.14 (m, 4H),
3.85 (dd J = 13.2, 2.3 Hz), 1.25 (t, J = 7.1 Hz, 3H), 1.11–1.00 (m, 28H),
0.03 (d, J = 14.2 Hz, 1H), −0.09 (d, J = 14.2 Hz, 1H); 13C NMR (400 MHz,
CDCl3) δ 170.0, 159.4, 147.9, 145.0, 138.8, 125.3, 88.5, 82.3, 81.1, 70.0, 68.0,
60.9, 60.1, 17.9, 17.7, 17.6, 17.6, 17.6, 17.5, 17.5, 14.3, 14.2, 14.1, 14.1, 13.9,
−9.91; Mass [MH]+ 595; Anal. Calcd for C27H46N4O7Si2: C, 54.52; H, 7.79;
N, 9.42. Found: C, 54.29; H, 7.51; N, 9.73.
2ꢁ-O-Carboxymethylinosine (1). To a solution of 4 (100 mg, 0.17
mmol) in anhydrous tetrahydrofuran (10 mL) was added TBAF (1 M
solution in tetrahydrofuran, 0.25 mL, 0.25 mmol) and the resulting mixture
was stirred at rt for 27 h, then concentrated under reduced pressure.
The resulting crude was purified by preparative TLC, eluting with 3:17
methanol/dichloromethane and extracting the silica gel with 1:19 to 1:13
methanol/dichloromethane, to give 1 as a yellow syrup (49 mg, 89%): Rf
24
0.30 (3:17 methanol/dichloromethane); [α]D −56.50 (c 0.31, CH3OH);
UV (λmax = 244.0 nm, 275.0 nm (sh), CH3OH); 1H NMR (400 MHz, DMSO-
d6): δ 12.44 (br s, 1H, D2O exchangeable), 8.34 (s, 1H), 8.08 (s, 1H), 6.03
(d, J = 6.0 Hz, 1H, anomeric), 5.34 (d, J = 4.9 Hz, 1H, D2O exchangeable),
5.12 (br t, J = 5.4 Hz, 1H, D2O exchangeable), 4.56 (dd, J = 6.0, 5.0 Hz,

232
F. Casu et al.
1H), 4.33 (dd, J = 8.2, 4.9 Hz, 1H), 4.27 (d, J = 16.8 Hz, 1H), 4.18 (d, J =
16.8 Hz, 1H), 3.99–3.96 (m, 1H), 3.62 (ddd, J = 12.1, 5.1, 4.2 Hz, 1H), 3.50
(dd J = 12.1, 5.7, 4.1 Hz, 1H); 13C NMR (400 MHz, CDCl3) δ 169.9, 156.6,
148.3, 146.0, 138.8, 124.4, 86.2, 85.4, 81.6, 68.7, 66.8, 60.6; Mass [MH]+ 327;
Anal. Calcd for C28H49N5O7: C, 44.18; H, 4.33; N, 17.17. Found: C, 44.46; H,
4.59; N, 17.33.
O6-Benzylinosine (8). A solution of 6-chloropurine riboside (2.0 g,
6.98 mmol) in anhydrous dimethylformamide (50 mL) was treated with
sodium benzyloxide (1 M solution in benzyl alcohol) at rt. After 5 h, the
reaction was diluted with water (300 mL) and the resulting mixture
was extracted with dichloromethane (3 × 100 mL). The combined or-
ganic phases were dried over magnesium sulfate, filtered, and concen-
trated under reduced pressure. The resulting crude was purified by TLC-
grade silica gel flash chromatography (dichloromethane to 1:32 methanol/
dichloromethane) to give 8 as an off-white solid (1.56 g, yield: 62%): Rf 0.33
25
(1:9 methanol/dichloromethane); mp 170–171◦C; [α]D −45.54 (c 0.36,
1
CH3OH); UV (λmax = 253.5 nm, 277 nm (sh), CH3OH); H NMR (400
MHz, DMSO-d6): δ 8.65 (s, 1H), 8.58 (s, 1H), 7.52–7.50 (m, 2H), 7.44–7.32
(m, 3H), 6.00 (d, J = 5.8 Hz, 1H, anomeric), 5.64 (s, 2H), 5.54 (d, J = 6.0
Hz, 1H, D2O exchangeable), 5.27 (d, J = 4.9 Hz, 1H, D2O exchangeable),
5.17 (t, J = 5.6 Hz, 1H, D2O exchangeable), 4.60 (dd, J = 11.0, 5.7 Hz, 1H),
4.17 (dd, J = 8.6, 4.9 Hz, 1H), 3.97 (dd, J = 7.5, 3.8 Hz, 1H), 3.72–3.60
(m, 1H), 3.60–3.53 (m, 1H); 13C NMR (400 MHz, DMSO-d6) δ 159.9, 152.0,
151.7, 142.6, 136.3, 128.6, 128.4, 128.3, 121.2, 87.8, 85.8, 73.8, 70.4, 67.9,
61.4; Mass [M + Na]+ 381; Anal. Calcd for C17H18N4O5: C, 56.98; H, 5.06;
N, 15.63. Found: C, 57.04; H, 5.16; N, 15.42.
3ꢁ,5ꢁ-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-O6-benzylinosine (9).
1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (1.06 mL, 3.32 mmol) was
added to a solution of 8 (1.08 g, 3.01 mmol) in anhydrous pyridine (40
mL), and the resulting solution was stirred at rt for 4 h. The solution was
then concentrated in vacuo and the residue purified by TLC-grade silica
gel flash chromatography (1:3 ethyl acetate/hexanes) to give 9 as a white
solid (1.62 g, yield: 90%): Rf 0.40 (1:19 methanol/dichloromethane); mp
26
48–50◦C; [α]D −27.38 (c 0.38, CHCl3); UV (λmax = 248.5 nm, CH3OH);
1H NMR (400 MHz, CDCl3): δ 8.49 (s, 1H), 8.08 (s, 1H), 7.54–7.52 (m, 2H),
7.37–7.30 (m, 3H), 6.02 (s, 1H, anomeric), 5.67 (s, 2H), 5.08 (dd J = 7.7,
5.7 Hz, 1H), 4.55 (d, J = 5.3 Hz, 1H), 4.18–4.00 (m, 3H), 3.22 (s, 1H, D2O
exchangeable), 1.20–1.00 (m, 28H); 13C NMR (400 MHz, CDCl3) δ 160.5,
152.0, 151.2, 141.1, 136.0, 128.4, 128.3, 128.1, 122.4, 89.7, 82.1, 75.1, 70.6,
68.4, 61.5, 17.4, 17.3, 17.3, 17.2, 17.1, 17.0, 16.9, 16.9, 13.2, 13.0, 12.7, 12.5;
Mass [MH]+ 601; Anal. Calcd for C29H44N4O6Si2: C, 57.97; H, 7.38; N, 9.32.
Found: C, 58.07; H, 7.38; N, 9.15.
3ꢁ,5ꢁ-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-2ꢁ-O-succinyl-O6-
benzylinosine (10). 4-(Dimethylamino)pyridine and succinic anhydride

Synthesis of 2 ꢁ-Substituted Inosine Analogs
233
were sequentially added to a stirred solution of 9 (0.31 g, 0.516 mmol) in
anhydrous pyridine (4 mL). The solution was stirred at rt for 6 h, then
quenched with water (20 mL) and extracted with ethyl acetate (3 × 10 mL).
The combined organic extracts were washed with brine (10 mL), dried over
magnesium sulfate, filtered, and concentrated under reduced pressure. The
resulting crude was purified by TLC-grade silica gel flash chromatography
(0 to 4% methanol/dichloromethane) to give 10 as an off-white solid (0.32
24
g, yield: 93%): mp 68–70◦C; [α]D −27.81 (c 0.44, CHCl3); UV (λmax
=
1
247.5 nm, CH3OH). H NMR (400 MHz, CDCl3): δ 9.75 (br s, 1H, D2O
exchangeable), δ 8.48 (s, 1H), 8.18 (s, 1H), 7.53–7.50 (m, 2H), 7.38–7.30
(m, 3H), 6.06 (s, 1H, anomeric), 5.79 (d, J = 5.3 Hz, 1H), 5.66 (s, 2H), 5.12
(dd, J = 8.9, 5.4, Hz, 1H), 4.18–3.90 (m, 3H), 2.80–2.60 (m, 4H), 1.11–0.90
(m, 28H); 13C NMR (400 MHz, CDCl3) δ 176.3, 171.2, 160.7, 152.5, 151.1,
141.8, 136.1, 128.6, 128.3, 122.0, 88.0, 82.2, 75.9, 69.0, 68.7, 60.5, 29.9, 29.2,
17.6, 17.6, 17.5, 17.2, 17.1, 13.5, 13.1, 13.0, 12.8; Mass [MH]+ 701; Anal.
Calcd for C33H48N4O9Si2: C, 56.55; H, 6.90; N, 7.99. Found: C, 56.55; H,
6.96; N, 7.68.
2ꢁ-O-Succinyl-O6-benzylinosine (11). To a stirred solution of 10 (0.17
g, 0.24 mmol) in anhydrous tetrahydrofuran (10 mL), triethylamine trihy-
drofluoride 0.20 mL, 1.24 mmol) was added and the resulting solution was
stirred at rt for 4 h. The solution was concentrated in vacuo and the residue
was dissolved in dichloromethane, loaded onto a TLC-grade column and
eluted with 2–4% methanol/dichloromethaneto give 11 as a white solid (90
23
mg, yield: 82%): mp 83–85◦C; [α]D −88.28 (c 0.48 CHCl3); UV (λmax
=
249.5 nm, CH3OH). 1H NMR (400 MHz, CDCl3): δ 8.48 (s, 1H), 8.23 (s, 1H),
7.53–7.49 (m, 2H), 7.38–7.29 (m, 3H), 5.95 (d, J = 7.0 Hz, 1H, anomeric),
5.68–5.57 (m, 5H), 5.09 (t, J = 5.9 Hz, 1H), 4.30 (s, 1H), 3.95 (d, J = 12.7
Hz, 1H), 3.81 (d, J = 12.7 Hz, 1H), 3.07 (t, J = 7.0 Hz, 6H), 2.76–2.65 (m,
4H), 1.27 (t, J = 7.0 Hz, 9H); 13C NMR (400MHz, CDCl3) δ 177.0, 171.9,
160.3, 151.2, 150.4, 143.1, 135.5, 128.2, 128.1, 128.0, 122.4, 90.6, 85.1, 74.2,
72.9, 68.4, 62.5, 30.1, 29.4; Mass [MH]+ 459; Anal. Calcd for C21H22N4O8:
C, 55.02; H, 4.84; N, 12.22. Found: C, 54.66; H, 5.22; N, 12.51.
2ꢁ-O-Succinylinosine (2). To a stirred solution of 11 (1.10 g, 2.40 mmol)
in ethanol (40 mL), 20% Pd(OH)2 (170 mg, 0.24 mmol) was added and the
resulting suspension was stirred under hydrogen at rt for 7 h. The mixture
was then filtered through a pad of Celite washing with ethanol and con-
centrated under reduced pressure to give 2 as a white solid (790 mg, yield:
23
88%): mp 109–111◦C (dec); [α]D −41.29 (c 0.50 CH3OH); UV (λmax
=
244.5 nm, 270.0 nm (sh), CH3OH). 1H NMR (400 MHz, DMSO-d6): δ 12.44
(br s, 1H, D2O exchangeable), 8.36 (s, 1H), 8.10 (s, 1H), 5.88 (d, J = 7.0
Hz, 1H, anomeric), 5.25 (dd, J = 5.3, 2.3 Hz, 1H), 4.78 (dd, J = 6.8, 5.5
Hz, 1H), 4.10 (dd, J = 6.1, 3.5 Hz, 1H), 3.66 (dd, J = 12.2, 3.7 Hz, 1H),
3.59 (dd, J = 12.0, 3.7 Hz, 1H), 2.65–2.60 (m, 2H), 2.55–2.49 (m, 2H); 13
C
NMR (400 MHz, D2O) δ 178.4, 174.3, 158.1, 148.2, 146.2, 140.4, 124.1, 88.3,

234
F. Casu et al.
83.9, 73.4, 72.8, 61.4, 30.0, 29.6; Mass [MH]+369; [M-H]− 367; Anal. Calcd
.
for C14H16N4O8 H2O: C, 43.53; H, 4.70; N, 14.50. Found: C, 43.73; H, 5.06;
N, 14.18.
REFERENCES
1. (a) Casu, F.; Chiacchio, M.A.; Romeo, R.; Gumina, G. Chiral synthesis of heterosubstituted nucleoside
analogs from non-carbohydrate precursors. Curr. Org. Chem. 2007, 11, 1017–1032; (b) Casu, F.;
Chiacchio, M.A.; Romeo, R.; Gumina, G. Chiral synthesis of carbocyclic nucleoside analogs from
non-carbohydrate precursors. Curr. Org. Chem. 2007, 11, 999–1016; (c) Gumina, G.; Olgen, S.; Chu,
C.K. Chiral synthesis of antiviral nucleosides from carbohydrate templates, in Antiviral Nucleosides:
Chiral Synthesis and Chemotherapy, ed. C.K. Chu, Elsevier Science, Atlanta, GA, 2003, pp. 77–189.
2. Gumina, G.; Choi, Y.; Chu, C.K. Recent advances in antiviral nucleosides, in Antiviral Nucleosides:
Chiral Synthesis and Chemotherapy, ed. C.K. Chu, Elsevier Science, Atlanta, GA, 2003, pp. 1–76.
3. (a) Legrand-Abravanel, F.; Nicot, F.; Izopet, J. New NS5B polymerase inhibitors for hepatitis C. Expert
Opin. Investig. Drugs 2010, 19, 963–975; (b) Carroll, S.S.; Olsen, D.B. Nucleoside analog inhibitors of
hepatitis C virus replication. Infect. Disord. Drug Targets 2006, 6, 17–29.
4. (a) Vorbru¨ggen, H.; Krolikiewicz, K.; Niedballa, U. Synthesis of nucleosides with use of trimethylsilyl-
heterocycles. Ann. N.Y. Acad. Sci. 1975, 255, 82–90; (b) Vorbru¨ggen, H.; Bennua, B. New simplified
nucleoside synthesis. Tetrahedron Lett. 1978, 19, 1339–1342.
5. (a) Miller, P.S.; TS’O, P.O.P. Linkage of proteins to nucleic acids. Patent No. US 4587044, May 1986;
(b) Sauer, C.; Schwabe, U. Method for the selective 2ꢁ-O-substitution of nucleosides. Z. Naturforsch.
C: J. Biosci. 1980, 35C, 163–167.
6. (a) Pierra, C.; Benzaria, S.; Amador, A.; Moussa, A.; Mathieu, S.; Storer, R.; Gosselin, G. NM 283,
an efficient prodrug of the potent anti-HCV agent 2ꢁ-C-methylcytidine. Nucleosides Nucleotides Nucleic
Acids 2005, 24, 767–770; (b) Eldrup, A.B.; Allerson, C.R.; Bennett, C.F.; Bera, S.; Bhat, B.; Bhat,
N.; Bosserman, M.R.; Brooks, J.; Burlein, C.; Carroll, S.S.; Cook, P.D.; Getty, K.L.; MacCoss, M.;
McMasters, D.R.; Olsen, D.B.; Prakash, T.P.; Prhavc, M.; Song, Q.L.; Tomassini, J.E.; Xia, J. Structure-
activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent
RNA polymerase. J. Med. Chem. 2004, 47, 2283–2295; (c) Carroll, S.; Tomassini, J.E.; Bosserman, M.;
Getty, K.; Stahlhut, M.W.; Eldrup, A.B.; Bhat, B.; Hall, D.; Simcoe, A.L.; LaFemina, R.; Rutkowski,
C.A.; Wolanski, B.; Yang, Z.; Migliaccio, G.; De Francesco, R.; Kuo, L.C.; MacCoss, M.; Olsen, D.B.
Inhibition of hepatitis C virus RNA replication by 2ꢁ-modified nucleoside analogs. J. Biol. Chem. 2003,
278, 11979–11984;
7. (a) Gundlach IV, C.W.; Ryder, T.R.; Glick, G.D. Synthesis of guanosine analogs bearing pendant
alkylthiol tethers. Tetrahedron Lett. 1997, 38, 4039–4042; (b) Grøtli, M.; Douglas, M.; Beijer, B.;
Garc´ıa, R.G.; Eritja, R.; Sproat, B. Protection of the guanine residue during synthesis of 2ꢁ-O-
alkylguanosine derivatives. J. Chem. Soc. Perkin Trans. 1 1997, 18, 2779–2786; (c) Markiewicz, W.T.
Tetraisopropyldisiloxane-1,3-diyl, a group for simultaneous protection of 3ꢁ- and 5ꢁ-hydroxy functions
of nucleosides. J. Chem. Res. Synop. 1979, 24–25; (e) Beijer, B.; Grøtli, M.; Douglas, M.E.; Sproat, B.S.
Simplified and cost effective syntheses of fully protected phosphoramidite monomers suitable for the
assembly of oligo(2ꢁ-O-allylribonucleotides). Nucleosides Nucleotides 1994, 13, 1905–1927; (f) Wada, T.;
Tobe, M.; Nagayama, T.; Furusawa, K.; Sekine, M. Regioselective protection of the 2ꢁ-hydroxyl group
of N-acyl-3ꢁ,5ꢁ-O-di(t-butyl) silanediylnucleoside derivatives by use of t-BuMgCl and 2-(trimethylsilyl)
ethoxymethyl chloride. Tetrahedron Lett. 1995, 36, 1683–1684; (g) Grøtli, M.; Douglas, M.; Beijer, B.;
Eritja, R.; Sproat, B. A simple method for the synthesis of 2ꢁ-O-alkylguanosine derivatives. Bioorg. Med.
Chem. Lett. 1997, 7, 425–428.
8. (a) Chow, S.; Wen, K.; Sanghvi, Y.S.; Theodorakis, E.A. MDPSCL2: A new protecting group for
chemoselective synthesis of 2ꢁ-O-alkylated guanosines. Nucleosides Nucleotides Nucleic Acids 2003,
22, 583–587; (b) Chow, S.; Wen, K.; Sanghvi, Y.S.; Theodorakis, E.A. Novel synthesis of 2ꢁ-O-
methylguanosine. Bioorg. Med. Chem. Lett. 2003, 13, 1631–1634; (c) Wen, K.; Chow, S.; Sanghvi, Y.S.;
Theodorakis, E.A. Synthesis of 2ꢁ-O-methoxyethylguanosine using a novel silicon-based protecting
group. J. Org. Chem. 2002, 67, 7887–7889;

Synthesis of 2 ꢁ-Substituted Inosine Analogs
235
9. (a) Haraguchi, K.; Nishikawa, A.; Sasakura, E.; Tanaka, H.; Nakamura, K.T.; Miyasaka, T. Electrophilic
addition to 4-thio furanoid glycal: A highly stereoselective entry to 2ꢁ-deoxy-4ꢁ-thio pyrimidine nu-
cleosides. Tetrahedron Lett. 1998, 39, 3713–3716; (b) Purdy, D.F.; Zintek, L.B.; Nair, V. Synthesis of
isonucleosides related to AZT and AZU. Nucleosides Nucleotides 1994, 13, 109–126.
10. Robins, M.J.; Samano, V.; Zhang, W.; Balzarini, J.; De Clercq, E.; Borchardt, R.T.; Lee, Y.; Yuan,
C.S. Nucleic acid related compounds. 74. Synthesis and biological activity of 2ꢁ(and 3ꢁ)-deoxy-
2ꢁ(and 3ꢁ)-methylenenucleoside analogs that function as mechanism-based inhibitors of S-adenosyl-
L-homocysteine hydrolase and/or ribonucleotide reductase. J. Med. Chem. 1992, 35, 2283–2293.
11. Nishino, S.; Takamura, H.; Ishido, Y. Regioselective protection of carbohydrate derivatives: Part. 20
simple, efficient 2ꢁ-o-deacylation of fully acylated purine and pyrimidine ribonucleosides through
tert-butoxide. Tetrahedron 1986, 42, 1995–2004.
12. (a) Bordon-Pallier, F.; Haesslein, J.–L. Process for preparation of new purine derivatives, their ap-
plication as drugs, pharmaceutical compositions containing them, and new uses for them. Periodic
No. FR 2851248, 20 August 2004; (b) Agarwal, A.; Chauhan, P. Convenient dimethylamino amina-
tion in heterocycles and aromatics with dimethylformamide. Synth. Commun. 2004, 34, 2925–2930;
(c) Abramov, M.; Marchand, A.; Calleja-Marchand, A.; Herdewijn, P. Synthesis of D-altritol nucle-
osides with a 3ꢁ-O-tert-butyldimethylsilyl protecting group. Nucleosides Nucleotides Nucleic Acids 2004,
23, 439–455; (d) Coppinger, G.M. Preparations of N ,N -dimethylamides. J. Am. Chem. Soc. 1954, 76,
1372–1373; (e) Deorha, D.S.; Sharma, H.L. Reaction of halonitrobenzenes with dimethylformamide.
J. Ind. Chem. Soc. 1963, 40, 819–820.
13. Liu, J.; Janeba, Z.; Robins, M.J. SNAr Iodination of 6-Chloropurine Nucleosides: Aromatic Finkelstein
reactions at temperatures below −40◦C. Org. Lett. 2004, 6, 2917–2919.