Synthesis and HCV inhibitory properties of 9-deaza- and 7,9-dideaza-7-oxa-2′-C-methyladenosine

Article abstract of DOI:10.1016/j.bmc.2007.05.020

As a part of an ongoing medicinal chemistry effort to identify inhibitors of the Hepatitis C Virus RNA replication, we report here the synthesis and biological evaluation of 9-deaza- and 7,9-dideaza-7-oxa-2′-C-methyladenosine. The parent 2′-C-methyladenos

Full text of DOI:10.1016/j.bmc.2007.05.020

Bioorganic & Medicinal Chemistry 15 (2007) 5219–5229
Synthesis and HCV inhibitory properties of 9-deaza-
and 7,9-dideaza-7-oxa-20-C-methyladenosine
Gabor Butora,a,* David B. Olsen,b Steven S. Carroll,b Daniel R. McMasters,a
Christoph Schmitt,a Joseph F. Leone,a Mark Stahlhut,b
Christine Burleinb and Malcolm MacCossa
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Antiviral Research, Merck Research Laboratories, West Point, PA 19486, USA
Received 8 February 2007; revised 4 May 2007; accepted 8 May 2007
Available online 22 May 2007
Abstract—As a part of an ongoing medicinal chemistry effort to identify inhibitors of the Hepatitis C Virus RNA replication, we
report here the synthesis and biological evaluation of 9-deaza- and 7,9-dideaza-7-oxa-20-C-methyladenosine. The parent 20-C-
methyladenosine shows excellent intracellular inhibitory activity but poor pharmacokinetic profile. Replacement of the nucleo-
side-defining 9-N of 20-C-methyladenosine with a carbon atom was designed to yield metabolically more stable C-nucleosides.
Modifications at position 7 were designed to exploit the importance of the hydrogen bond accepting properties of this heteroatom
in modulating the adenosine deaminase (ADA) mediated 6-N deamination. 7-Oxa-7,9-dideaza-20-C-methyladenosine was found to
be a moderately active inhibitor of intracellular HCV RNA replication, whereas 9-deaza- 20-C-methyladenosine showed only weak
activity despite excellent overlap of both of the synthesized target compounds with 20-C-methyladenosine’s three dimensional struc-
ture. Position 7 of the nucleobase proved to be an effective handle for modulating ADA-mediated degradation, with the rate of deg-
radation correlating with the hydrogen-bonding properties at this position.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Modifications in the vicinity of the 30-hydroxyl of the ri-
bose in natural ribonucleosides can produce effective
RNA chain terminators.8 For example, replacement of
the 20-hydrogen of natural ribonucleosides with a methyl
group yields compounds with excellent chain-terminating
properties.9 20-C-methyladenosine (1) (Fig. 1) was found
to inhibit HCV RNA replication without significant tox-
icity.9,10 Unfortunately, the poor pharmacokinetic
Hepatitis C virus (HCV) infection is a leading cause of
chronic hepatitis, liver cirrhosis, and hepatocellular car-
cinoma.1 At the present time the therapeutic options are
limited to subcutaneous administration of often poorly
tolerated pegylated interferon-a in combination with
the orally dosed nucleoside analog ribavirin.2,3
Recent advances in the molecular virology of HCV4
have led to the identification of a number of antiviral
molecular targets such as interference with viral poly-
peptide processing, RNA regulatory elements, and
RNA replication.5,6 Valopicitabine (NM283), currently
in development by Idenix, is an example of an NS5B
RNA-dependent RNA polymerase inhibitor.7
HO
HO
N
O
O
NH2
NH2
N
N
N
N
N
N
N
HO
OH
HO
HO
OH
1
2
HO
HO
NH
O
O
O
NH2
NH2
N
N
N
HO
OH
OH
Keywords: Nucleosides; Hepatitis C; Hepatitis C virus; RNA poly-
merase; Adenosine deaminase.
3
4
*
Corresponding author. Tel.: +1 732 594 8563; fax: +1 732 594
9556; e-mail: gabor_butora@merck.com
Figure 1. C02-Methyl adenosines.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.020

5220
G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
TolO
properties (mainly its rapid metabolic degradation) se-
verely limit its potential therapeutic use. Systematic inves-
tigation in this series has shown that replacement of the
7-N of the heterobase with a carbon atom (20-C-methyl-
7-deaza-adenosine, 2, Fig. 1) yields a compound with
even higher potency and much improved pharmacoki-
netic properties.11,12 The absence of 7-N greatly contrib-
utes to its metabolic stability, as the major pathway of
adenosine degradation, 6-NH2 deamination, requires
the presence of a hydrogen bond accepting heteroatom
at position 7 of the heterobase.13
TolO
ia or ib
HO
O
O
O
OH
OR
OR
ii
TolO
TolO
HO
OH
OH
OH
5
6, R = Me
7, R = Me
12, R = Allyl
11, R = Allyl
iii
TrO
HO
TrO
O
O
O
OR
OH
OR
iv
v or vi
O
O
O
O
O
O
In order to obtain further structure–activity informa-
tion, 20-C-methyl-9-deaza-adenosine (3, Fig. 1), a struc-
tural isomer of 2, was synthesized. The replacement of
9-N with a carbon atom should stabilize the molecule
and for tautomeric reasons the character of the 7-N
changes from a hydrogen bond acceptor to donor. Gi-
ven the role 7-N plays in 6-NH2 deamination,13 20-C-
methyl-9-deaza-adenosine should be metabolically more
stable than 20-C-methyl-adenosine (1). In the case of 20-
C-methyl-7,9-dideaza-7-oxa-adenosine (4, Fig. 1), the
replacement of 9-N for carbon was designed to stabilize
the molecule, while the swap of 7-N for oxygen changes
the character of the 7-heteroatom from donor to an a
hydrogen bond acceptor.
10
9, R = Me
14, R = Allyl
8, R = Me
13, R = Allyl
Scheme 1. Reagents and conditions: (i) a—methanol or b—allyl
alcohol, PPTSA, 100 ꢁC, 24 h; (ii) MeONa, MeOH, rt, 2 h; (iii) 2,2-
dimethoxypropane, pTSA, rt, 2 h; (iv) triphenylchloromethane, Py,
60 ꢁC, 18 h; (v) R = Me; a—AcOH, Ac2O, DCM, cat.H2SO4, 0 ꢁC,
18 h; b—aq. NaOH, 15 min; (vi) R = Allyl, 1,3-bis(diphenylphosphi-
no)propane nickel (II) dichloride, Dibal-H, diethyl ether, 0 ꢁC, then rt
60 min.
approaches for effective removal of the 10-O-allyl pro-
tecting group in 14 were tested,23–25 and the most satis-
factory procedure was found to be the nickel (II)-
catalyzed Dibal-H-mediated reductive cleavage devel-
oped by Taniguchi.26
2. Chemistry
When 20-C-methyl-20,30-O-(1-methylethylidene)-50-O-
trityl-D-ribofuranose (10) was subjected to the Moffatt
procedure,25 intermediates 15 and 16 were obtained in
excellent yield (Scheme 2). The observed diastereoiso-
Although published procedures14,15 for preparation of
substituted 9-deazaadenosine analogs allowed for a di-
rect glycosylation of a suitably substituted heterocycle
(e.g., guanine or 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimi-
dine) under Vorbruggen conditions, a well-established
and more flexible stepwise approach was followed.16,17
TTO
TTO
TTO
O
O
O
OH
CN
CN
i
30,50-O-(4-Methylbenzoyl)-20-C-methyl-ribose (5) could
be conveniently synthesized following procedure relying
on a base-catalyzed intramolecular ester migration in
acylated carbohydrates.18,19 Our initial attempts at 10-
C-functionalization of 20,30,50-(4-methylbenzoyl)-pro-
tected ribose failed and a synthetic sequence yielding
20-C-methyl ribose equipped with base-stable protecting
groups had to be developed, Scheme 1.
+
O
O
O
O
O
O
10
15
16
ii
H
N
OH
CN
TTO
CN
iii
TTO
O
O
CN
According to this sequence, a pyridinium para-toluene-
sulfonate catalyzed glycosylation of 5 performed ini-
tially with methyl alcohol afforded the glycoside 6. The
ester groups were removed by a sodium methoxide cat-
alyzed trans-esterification, the vicinal 20,30-diol in 7 con-
verted to an acetonide (8), and the 50-primary hydroxyl
was protected with a trityl group (9). Even though the
usual conditions used to cleave glycosides (such as de-
scribed in ref. 20) followed by base hydrolysis did pro-
duce the desired hemiacetal 10, a partial cleavage of
the 50-O-trityl group induced by the acidic conditions
rendered the reaction poorly reproducible. Therefore,
the 10-O-methyl group was replaced with a group the
removal of which does not require the scission of the
glycosidic 10-C–O bond, such as allyl.21,22 The above se-
quence could be easily modified (intermediates 11–14) to
yield the respective 10-O-allyl glycoside 14. A number of
O
O
O
O
17
18
iv
O
N
O
OEt
OEt
CN
TTO
TTO
N
CN
O
O
v
CN
NH2
O
O
O
O
19
20, 1'-C(R)
21, 1'-C(S)
Scheme 2. Reagents and conditions: (i) diethyl cyanomethylphospho-
rane, NaH, DME, 0 ꢁC then rt, 4 h; (ii) LDA, MeOCHO, THF,
ꢀ78 ꢁC, 2 h then rt 20 min; (iii) H2NCH2CN, NaOAc, MeOH, rt, 72 h;
(iv) EtOCOCl, DBU, DCM, rt; (v) DBU, DCM, rt, 24 h.

G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
5221
CN
O
meric ratio of approximately 1–2 was in excellent agree-
H2N
OR
ment with that reported in the case of ribose.27 The 10-C
chiral center established during the Moffatt procedure
was expected to be isomerized at the subsequent step;
therefore the choice of isomer 16 over 15 for further
manipulations was arbitrary. The hydroxymethylene
group in 17 can be introduced following the known28
two-step procedure, but we found that this could be
more readily accomplished by a direct Claisen condensa-
tion. Indeed, the carbanion derived from 16 reacted
smoothly with methyl formate, and the pyrrole ring
was easily constructed following a published16 DBU-in-
duced Kirsch type29 cyclization. After the formation of
the enamine 18, the system was stabilized by N-acylation
(19) which also greatly facilitated the DBU-induced
cyclization. The isomeric pyrroles 20 and 21 (produced
in a ratio of 1:5) were separated by column chromato-
graphy, and the stereochemistry at 10-C was established
by nOe experiments (isomers 22 and 24).
TTO
TTO
iii
TTO
i
O
O
O
CN
CN
O
O
O
O
O
O
15
17, 1'-C(R), R=H
27, 1'-C(S), R=CH2CN
28, 1'-C(R)
29, 1'-C(S)
ii
iv
N
N
N
NH2
N
NH2
TTO
HO
O
O
O
v
O
O
O
HO
OH
4, 1'-C(R)
32, 1'-C(S)
30, 1'-C(R)
31, 1'-C(S)
Scheme 4. Reagents and conditions: (i) LDA, MeOCHO, THF,
ꢀ78 ꢁC, 2 h then rt 20 min; (ii) ClCH2CN, Cs2CO3, DMF, rt, 18 h;
(iii) LDA, ꢀ78 ꢁC, 2 h; (iv) formamidine acetate, EtOH, 85 ꢁC, 12 h;
(v) HCl, dioxane, MeOH, rt, 2 h.
The ethoxycarbonyl protecting group in 20 was hydro-
lyzed (22), and the pyrimidine ring was constructed fol-
lowing a published procedure.16 The trityl and acetonide
protecting groups in 23 were removed under acidic con-
ditions to provide nucleoside 3. A similar sequence was
used to obtain the 10-C-a isomer 26 (Scheme 3).
3. Methods
The ability of the nucleoside analogs to inhibit HCV
RNA replication was assessed using the cell-based bicis-
tronic replicon assay30 as modified for RNA quantita-
tion by RNase protection.9 In the replicon assay the
intracellular levels of replicon RNA are maintained by
the activity of the virally encoded replication complex.
Control assays titrating the inhibitory potency (EC50 va-
lue) of compounds 1 and 2 demonstrated results that
were similar to previous work.9,11
Intermediates 15 or 16 served as a convenient starting
point17 for the synthesis of the furan analog 4 (Scheme
4). The arbitrarily chosen b-isomer 15 was subjected to
a Claisen condensation with methyl formate yielding
17 as a mixture of diastereoisomers. O-Alkylation of
the enol 17 with chloroacetonitrile smoothly produced
the enol ether 27. In the absence of the stabilizing effect
of the ethoxyurethane group, such as in 19, the Kirsch
cyclization was much less efficient, and the desired fur-
ans 28 and 29 were obtained in a combined yield of only
12%. The respective a- and b-isomers were separated,
and the pyrimidine ring was constructed with formami-
dine. A one-pot acid-catalyzed cleavage of the protect-
ing groups then completed the synthesis of nucleosides
4 and 32.
The ability of adenosine deaminase (ADA) to convert
compounds 1–4 to the corresponding 6-oxo analogs
was determined in vitro. Two different reaction condi-
tions were investigated.12 The milder reaction condition
I was developed that would only partially convert 1 to
20-C-methyl-inosine. Reaction condition II contained a
higher enzyme concentration, higher reaction tempera-
ture, and longer reaction times, to differentiate whether
the nucleoside analog was a poor substrate or was stable
to ADA. Under reaction condition II, 1 was completely
converted to 20-C-methyl-inosine.
H2N
H2N
CN
CN
NH
TTO
TTO
O
O
OEt
N
i
O
O
O
O
O
Computer-aided conformational analyses were per-
formed as described previously.12 For analysis of elec-
trostatic potentials, the compounds were optimized at
the B3LYP/6-31G** level using Gaussian03.31 Electro-
static potential was plotted on the electronic density sur-
face at the B3LYP/6-31G** level using Spartan ’04
version 1.0.3.32
22, 1'-C(R)
24, 1'-C(S)
20, 1'-C(R)
21, 1'-C(S)
N
ii
N
N
NH2
N
NH2
TTO
HO
O
NH
O
NH
iii
O
O
HO
OH
4. Results and discussion
3, 1'-C(R)
23, 1'-C(R)
25, 1'-C(S)
Our approach to finding NS5B polymerase nucleoside
inhibitors with altered susceptibility to ADA-mediated
metabolism was to vary the base in such way as to mod-
ulate the electrostatic potential at position 7 without
26, 1'-C(S)
Scheme 3. Reagents and conditions: (i) K2CO3, EtOH, rt, 1 h; (ii)
formamidine acetate, EtOH, 90 ꢁC, 5 h; (iii) HCl, dioxane, rt, 24 h.

5222
G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
Table 1. Inhibitory activity of nucleoside analogs in the cell-based
a
replicon assay
changing the overall shape and conformation of the
molecule, thereby maximizing the probability of the
nucleosides being recognized by the appropriate kinases
and by NS5B polymerase. The conformational prefer-
ences of 1–4 were calculated using the MMFFs force
field and a dielectric constant of 50. The variations to
the base in 1–4 were found to have no profound effect
on the molecules’ calculated conformational prefer-
ences. In each case, the lowest-energy conformation
was found to be that with the ribofuranose ring in a
Northern pucker induced by the 20-C-Me group with a
pseudorotational angle P of 8.5–16.4 and pucker ampli-
tude s of 38.9–39.2, and with the glycosyl torsion angle
in the anti-configuration. The calculated energy differ-
ence between Northern and Southern puckers differed
little among the four compounds, with the Northern
conformation preferred by 2.3–2.5 kcal/mol. The syn–
anti energy difference varied somewhat, from 0.57 kcal/
mol for compound 2 to 0.14 kcal/mol for 4. Superposi-
tion of the lowest energy conformations shows very
good alignment of the sugar rings with only slight vari-
ation in the base due to a change in bond angles in the
base upon modification of 9-N to carbon (Fig. 2).
c
Compound
EC50 (lM) replicon assay
CC50 (lM)
24 hb
72 hb
1
0.25
0.3
0.2 (n = 3)
0.11 (n = 3)
59.7 (n = 2)
8.1 (n = 3)
1.50 (n = 2)
>100
>100
>100
>100
>100
2
3
>100
22 (n = 3)
7.45 (n = 1)
4
Valopicitabine
a Compound inhibitory potency was determined in Huh-7 cells har-
boring a neomycinR-encoding replicon from HCV genotype 1b using
an RNase protection assay, as described in Section 3.
b
Length of time that the compound was incubated in the presence of
replicon-containing cells, prior to the determination of viral RNA
levels.
c Compound cytotoxicity as determined using an MTS assay, as
described in Section 3.
on several assumptions: (a) the main degradation path-
way of these compounds is 6-NH2 deamination,33 (b)
the hydrogen-bond acceptor at base position 7 is critical
for recognition by the protonated Asp296 of adenosine
deaminase (ADA)34, and (c) the capacity of the 7-het-
eroatom to accept a hydrogen bond is determined by
the electron density at this position.
Given the excellent conformational overlap between
highly active nucleosides 1 or 2 and the 9-deaza nucleo-
side 3, we were surprised to find out that the pyrrole
analog 3 displayed only rather weak anti-HCV activity
(EC50 59.7 lM, 72 h) when evaluated in the replicon as-
say. On the other hand, the furan analog 4 was moder-
ately active (EC50 22 lM) in a 24-h assay and showed
about threefold improved potency (EC50 8.1 lM) when
incubated in the presence of replicon-containing cells
for 72 h. The results are summarized in Table 1.
To gauge the effect of variation in the base on electronic
properties, ab initio calculations were performed on the
9-Me-substituted bases of 1–4 at the B3LYP/6-31**
level. Inclusion of the ribose did not change the results
substantially. Plots depicting the B3LYP/6-31G** elec-
trostatic potential mapped onto the molecular electronic
density are presented in Figure 3. The naturally occur-
ring adenine base of 1 is much more electronegative at
the 7-position relative to the furan 4, suggesting that
the furan will be a poorer hydrogen bond acceptor than
adenine. These results are in good agreement with the
findings of Nobeli et al.,35 who calculated that furan is
a much weaker H-bond acceptor than pyridine, due
largely to its lower negative partial charge at the accep-
tor atom. Calculation of pyridine and furan at the same
level of theory as used to calculate 1–4 gave similar
partial charges as those found on 7-N of 1 and 7-O of
4, respectively.
The apparent improvement of potency of the furan ana-
log 4 in the 72-h replicon assay likely reflects an increase
in the concentration of intracellularly generated 50-tri-
phosphate of 4. This observation suggests that the most
likely reason for low antiviral activity of 3 and 4 is the
slow conversion of the parent nucleosides to their
respective 50-triphosphates.
The design of metabolically more robust nucleosides
than adenosine or its 20-C-methyl analog 1 was based
Figure 2. Overlaid lowest-energy minima (MMFFs, e = 50) identified
for 1–4. Nitrogen atoms are shown in blue, oxygen atoms in red, and
carbon atoms are color-coded by molecule: 1 (green), 2 (gray), 3
(orange), and 4 (magenta).
Figure 3. Electron density surfaces of the 9-Me-substituted bases from
compounds 1–4 colored by the electrostatic potential (1, top left; 2, top
right; 3, bottom left; 4, bottom right; blue, electropositive; red,
electronegative; green, neutral).

G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
5223
Table 2. Adenosine deaminase-mediated conversion of nucleoside
analogsa
chemical shifts (ppm) are positive in the low field direc-
tion and were referenced indirectly through the solvent’s
residual signal to TMS. Elemental analyses were per-
formed at Robertson Microlit Laboratories, Madison
NJ.
Compound
Reaction conditions
Ib
Reaction conditions
IIc
5 lMd
25 lMd
5 lMd
25 lMd
1
2
3
4
14
0
9
0
0
0
100
0
100
0
5.1. Assay for inhibition of HCV RNA replication in cells
0
0
100
0
77
Inhibition of HCV RNA replication was assayed in a
subgenomic bicistronic replicon assay in HB-1 cells by
an in situ ribonuclease protection assay (RPA), as previ-
ously described after either 24- or 72-h incubation of the
compound with the cells.15 The replicon EC50 values are
the average of at least two separate determinations
(Table 1). The cytotoxicity of the nucleosides was deter-
mined by an MTS assay at 24 h.15
0
a Numbers represent the % of the analog converted to the corre-
sponding 6-oxo analog under the assay conditions.
b Reactions included 0.00625 U/mL ADA and were carried out at
30 ꢁC for 30 min. Under these conditions adenosine was 100% con-
verted to inosine.
c Reactions included 1.25 U/mL ADA and were carried out at 37 ꢁC
for 24 h. Under these conditions adenosine was 100% converted to
inosine.
d Concentration of the nucleoside analog in the reaction.
5.2. Assay for ADA mediated conversion
The observation that the hydrogen-bonding capacity at
position 7 is weaker in 4 than in 1 and lacking altogether
in 2 and 3 is consistent with the observed rates of deam-
ination by ADA presented in Table 2. Compounds 2–4
were completely stable to conversion under reaction
condition I, indicating that they are, at the least, less effi-
cient substrates for ADA, whereas 2 and 3 were com-
pletely stable. Compound 4 was converted to the
corresponding 6-oxo analog under reaction condition
II, suggesting that nucleoside 4 is a better substrate for
ADA compared to 2 and 3.
Assay reactions included 5 mM Tris–HCl, pH 7.4,
5 mM potassium phosphate, and 0.00625 or 1.25 U/
mL adenosine deaminase (ADA, Sigma A-5168, type
IX from calf spleen). Reactions were then initiated with
addition of 25 lM nucleoside. Reaction mixtures were
incubated at room temperature for 30 min (0.00625 U/
mL ADA) or 24 h (1.25 U/mL ADA) before the enzyme
was heat inactivated (75 ꢁC for 15 min). RP-HPLC ana-
lysis utilized a SUPELCOSIL LC-18-S column (Supelco
58931, 150· 4.6 mm, 5 lM) with 97.5%/2.5% 50 mM
potassium phosphate, pH 4.4/MeOH (Buffer A) and
80%/20% 50 mM potassium phosphate, pH 4.4/MeOH
(Buffer B), 1 mL/min flow rate. A 25 lL reaction volume
injection and elution over the gradient yielded quantifi-
able nucleoside peaks (0% B hold for 3 min, 0–100% B
over 9 min, 100% hold for 3 min, 10060% over
4.5 min) monitoring at 254 and 280 nm. Injections of
adenosine and inosine yielded linear standard curves
allowing for the quantitation of substrate utilization
and product formation.
In conclusion, novel 20-C-methyl 9-deaza nucleosides 3
and 4 were successfully synthesized and their basic phar-
macological properties evaluated. While the pyrrole
derivative 3 was only weakly active, the 7-oxa analog
4 showed moderate anti-HCV activity in the standard
24- and 72-h replicon assay. The apparent improvement
of the antiviral potency in the 72-h assay supports the
hypothesis of a gradual intracellular buildup of the cor-
responding nucleoside 50-O-triphosphates directly
responsible for the inhibition of the NS5B polymerase.
We have also demonstrated that the main degradation
pathway of adenosines, 6-N deamination, can be effec-
tively influenced by manipulation of electron density at
position 7 of the heterobase.
5.3. 10-O-Methyl 2-C-methyl-3,5-bis-O-(4-methylbenzoyl)-
a-D-ribofuranoside (6)
A solution of diol 5 (1.28 g, 3.19 mmol) and pyridinium
para-toluenesulfonate (360 mg, 1.43 mmol) in anhy-
drous MeOH (6 mL) was heated to 80 ꢁC in a sealed
tube for 24 h. The solvent was evaporated and the resi-
due was purified by flash chromatography (silica-gel,
EtOAc/hexanes EtOAc: 0–60%) to yield 977 mg (74%)
5. Experimental
1
All non-hydrolytic reactions, unless indicated otherwise,
were carried out in dry solvents purchased from Aldrich.
HPLC analyses were performed at ambient temperature
using a Waters XTerra C18, 5 lm, 50· 4.6 mm reversed
phase column under following conditions: flow rate:
1.25 mL/min; eluent A: water (0.1% TFA), Eluent B:
acetonitrile (0.1% TFA). Gradient: %B (time) starting
at 10 (0) 50 (3), 90 (9), 100 (10), 100 (13), detector:
UV at 220 nm. Analytical thin layer chromatography
was performed on Silica Gel 60 F254 plates, 5· 10 cm,
with a layer thickness of 250 lm and preparative TLC
on Analtech Silica Gel GF plates, 20· 20 cm, with a
layer thickness of 1000 lm. NMR spectra were recorded
on a Varian Inova 500 or 600 MHz spectrometer. The
of the desired product as a single diastereoisomer. H
NMR (500 MHz, CDCl3) d: 7.93 (d, J = 8.2 Hz, 2H),
7.90 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 7.14
(d, J = 8.2 Hz, 2H), 5.57 (d, J = 7.10 Hz, 1H), 4.73 (s,
1H), 4.58 (dd, J = 11.5, 3.9 Hz, 1H), 4.52 (m, 1H),
4.44 (dd, J = 11.7, 5.5 Hz, 1H), 3.37 (s, 3H), 2.41 (s,
3H), 2.36 (s, 3H), 1.37 (s, 3H). nOe (4 mg/mL, CDCl3):
irradiation of the singlet at 4.73 ppm (10-H) resulted in a
weak enhancement of the doublet at 5.57 ppm (30-H) by
<1%. 13C NMR (500 MHz, CDCl3) d: 166.29, 165.58,
144.40, 143.54, 129.81, 129.68, 129.20, 128.91, 127.08,
126.28, 108.98, 79.49, 78.33, 77.11, 65.18, 55.18, 21.64,
21.57, 19.90. Mp: 107–109 ꢁC. HRMS for C23H26O7 cal-
culated 437.1576 [M+Na]+, found: 437.1584. CHN

5224
G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
analysis: for C23H26NO7 calculated C, 66.65%; H,
6.32%; found: C, 66.68%; H, 6.54%.
was treated at 0 ꢁC with Dibal-H (2 mL, 1 M solution
in hexanes). Stirring at 0 ꢁC was continued for 30 min
and the reaction was quenched with 2 mL of water. It
was stirred at ambient temperature for an additional
30 min, anhydrous magnesium sulfate was added (ca.
2 g), and the stirring was continued for another 30 min.
The drying agent was filtered off, the solvent was evapo-
rated, and the residue (795 mg) was purified by gradient
chromatography (silica-gel, EtOAc/hexanes, EtOAc: 0–
70%) to afford 536 mg (73%) of the desired product as
mixture of epimers (ca. 1.2:1). 1H NMR (500 MHz,
CD3Cl) major isomer (assignment based on integration
and coupling pattern): d: 7.30–7.40 (m, 12H), 7.25–7.30
(m, 3H), 5.22 (d, J = 8.0 Hz, 1H), 4.43 (br s, 1H), 4.27
(t, J = 4.10 Hz, 1H), 3.57 (d, J = 7.80 Hz, 1H), 3.41
(dd, J = 10.3, 3.9 Hz, 1H), 3.14 (dd, J = 10.1, 3.7 Hz,
1H), 1.49 (s, 3H), 1.46 (s, 6H). Minor isomer: d: 7.30–
7.40 (m, 12H), 7.25–7.30 (m, 3H), 5.14 (d, J = 10.5 Hz,
1H), 4.32 (br s, 1H), 4.24 (t, J = 3.7 Hz, 1H), 3.84 (d,
J = 10.5 Hz, 1H), 3.35 (m, 2H), 1.56 (s, 3H), 1.46 (s,
6H). 13C NMR (500 MHz, CD3Cl) major isomer (assign-
ment based on peak height): 142.97, 128.73, 127.97,
127.38, 112.62, 104.40, 92.04, 87.56, 87.19, 85.22, 64.76,
28.44, 27.83, 20.30. Minor isomer: 143.46128.68,
127.88, 127.13, 113.54, 101.92, 88.00, 87.76, 87.37,
80.91, 64.24, 27.31, 27.25, 22.04. HRMS for C28H30O5
calculated 469.1991 [M+Na]+, found: 469.1987. CHN
analysis: for C28H30O5 calculated C, 75.31%; H, 6.77%;
found: C, 75.12%; H, 6.99%.
5.4. 10-O-Methyl-20-C-methyl-20,30-O-(1-methylethylide-
ne)-a-D-ribofuranoside (8)
A solution of the diester 6 (950 mg, 2.2922 mmol) in
MeOH (10 mL) was treated with a methanolic solution
of sodium methoxide (0.5 M, 3 mL), and stirring at
room temperature was continued overnight. The reac-
tion was quenched by addition of ethereal hydrogen
chloride (1 N, 1.5 mL), and the volatiles were evapo-
rated. The crude triol was dissolved in DCM (6 mL)
and 2,2-dimethoxypropane (6 mL) was added, followed
by a catalytic amount of para-toluenesulfonic acid. The
reaction mixture was stirred at room temperature for
30 min and quenched by pouring onto a saturated solu-
tion of sodium bicarbonate (10 mL). The product was
extracted with DCM (4· 30 mL), dried with anhydrous
sodium sulfate, and evaporated to dryness. Purification
by flash chromatography (silica-gel, EtOAc/hexanes
EtOAc: 0–70%) afforded 345 mg (69%, two steps) of
the desired product. 1H NMR (500 MHz, CDCl3) d:
4.81 (s, 1H), 4.44 (s, 1H), 4.26 (t, J = 3.0 Hz, 1H), 3.66
(dd, J = 12.4, 2.8 Hz, 1H), 3.58 (dd, J = 12.4, 3.2, 1H),
3.41 (s, 3H), 1.44 (s, 3H), 1.40 (s, 3H), 1.39 (s, 3H).
13C NMR (500 MHz, CDCl3) d: 112.40, 110.91, 92.00,
88.00, 87.30, 63.82, 55.86, 28.00, 27.57, 19.56. LCMS:
for C10H18O5 calculated 218.12, found: 241.30
[M+Na]+. CHN analysis: for C10H18O5 calculated C,
55.25%; H, 8.31%; found: C, 55.03%; H, 8.53%.
5.7. 10-O-Allyl-20-C-methyl-30,50-bis-O-(4-methylbenzoyl)-
D-ribofuranoside (11)
5.5. 10-O-Methyl-20-C-methyl-20,30-O-(1-methylethylid-
ene)-50-O-trityl-a-D-ribofuranoside (9)
A solution of 20-C-methyl-3,5-bis-O-(4-methylbenzoyl)-
D-ribofuranose (5) (2.00 g, 5.00 mmol) and pyridinium
para-toluenesulfonate (627 mg, 2.5 mmol) in allyl alco-
hol (10 mL) was heated to 100 ꢁC for 24 h. The volatile
allyl alcohol was evaporated, and the residue was puri-
fied by gradient column chromatography (silica-gel,
EtOAc/hexanes, EtOAc: 0–30%) to yield 3.55 g (81%)
A solution of alcohol 8 (612 mg, 2.80 mmol) in pyridine
was treated with trityl chloride (782 mg, 2.80 mmol) and
the reaction mixture was stirred at 60 ꢁC overnight.
It was poured into a 10% aqueous solution of citric
acid (20 mL), and the crude product was extracted
with tert-butyl methyl ether (3· 30 mL). The combined
extracts were dried with anhydrous magnesium sulfate,
filtered, and evaporated to dryness. The residue
(1.3747 g) was purified by flash chromatography (silica-
gel, EtOAc/hexanes, EtOAc: 0–30%) to yield 917 mg
(71%) of the pure product. 1H NMR (500 MHz, CDCl3)
d: 7.50 (m, 6H), 7.32 (m, 6H), 7.27 (m, 3H), 4.78 (s, 1H),
4.32 (dd, J = 8.0, 6.2 Hz, 1H), 4.23 (s, 1H), 3.27 (dd,
J = 8.4, 6.0 Hz, 1H), 3.19 (s, 3H), 3.12 (t, J = 8.9 Hz,
1H), 1.51 (s, 3H), 1.44 (s, 3H), 1.25 (s, 3H). 13C NMR
(500 MHz, CDCl3) d: 143.82, 128.60, 127.90, 127.78,
127.23, 127.02, 112.47, 110.62, 91.21, 88.23, 86.67,
84.64, 64.04, 55.11, 28.09, 27.71, 20.40. HRMS for
C29H32O5 calculated 483.2148 [M+Na]+, found:
483.2132. CHN analysis: for C29H32O5 calculated C,
75.63%; H, 7.00%; found: C, 75.58%; H, 7.15%.
1
of the pure product. H NMR (500 MHz, CDCl3) d:
7.93 (d, J = 8.2 Hz, 2H), 7.88 (d, J = 8.0 Hz, 2H), 7.23
(d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 2H), 5.85 (m,
1H), 5.56 (d, J = 7.1 Hz, 1H), 5.26 (dd, J = 17.4,
1.8 Hz, 1H), 5.18 (dd, J = 10.5, 1.6 Hz, 1H), 4.88 (s,
1H), 4.43 to 4.60 (bm, 3H), 4.25 (ddt, J = 13.3, 5.0,
1.6 Hz, 1H), 4.0 (dt, J = 13.3, 5.7, 1.4 Hz, 1H), 2.42 (s,
3H), 2.28 (s, 3H), 1.41 (s, 3H). nOe (4 mg/mL, CDCl3):
irradiation of the singlet at 4.88 ppm (10-H) resulted in a
weak enhancement of the doublet at 5.56 ppm (30-H) by
<1%. 13C NMR (500 MHz, CDCl3) d: 166.29, 165.57,
144.41, 143.54, 133.76, 129.83, 129.73, 129.22, 128.93,
127.02, 126.28, 119.98, 107.01, 79.64, 78.32, 77.16,
68.23, 65.22, 21.66, 21.59, 20.00. Mp: 64 ꢁC. HRMS
for C25H28O7 calculated 463.1733 [M+Na]+, found:
463.1727. CHN analysis: for C25H28O7 calculated C,
68.17%; H, 6.41%; found: C, 68.15%; H, 6.43%.
5.6. 20-C-Methyl-20,30-O-(1-methylethylidene)-50-O-tri-
tyl-D-ribofuranose (10)
5.8. 10-O-Allyl-20-C-methyl-D-ribofuranoside (12)
A solution of the allylether 14 (800 mg, 1.6441 mmol)
and 1,3-bis(diphenylphosphino)propane nickel (II)
dichloride (27 mg, 0.05 mmol) in diethyl ether (16 mL)
A solution of diester 11 (3.55 g, 8.059 mmol) was dis-
solved in anhydrous MeOH (20 mL) and treated with
a methanolic solution of sodium methoxide (5 mL,

G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
5225
0.5 M solution). Stirring at room temperature was con-
tinued for 2 h, and the reaction was quenched by addi-
tion of 1N HCl (3 mL). The solvent was evaporated
and the residue was purified by column chromatography
(silica-gel, EtOAc, 100% EtOAc, isocratic) to obtain
1.1372 g (69%) of the desired product in the form of a
white powder. 1H NMR (500 MHz, CDCl3) d: 5.88
(m, 1H), 5.29 (dd, J = 17.2, 1.4 Hz, 1H), 5.20 (dd,
J = 10.3, 1.2 Hz, 1H), 4.80 (s, 1H), 4.23 (dd, J = 13.0,
5.0 Hz, 1H), 4.02 (m, 3H), 3.82 (dd, J = 11.7, 2.5 Hz,
1H), 3.66 (dd, J = 11.9, 3.7 Hz, 1H), 1.33 (s, 3H). 13C
NMR (500 MHz, CDCl3) d: 133.73, 117.46, 107.24,
83.64, 79.18, 74.86, 68.97, 62.97, 19.33. Mp: 66–71 ꢁC.
HRMS for C9H16O5 calculated 227.0895 [M+Na]+,
found: 227.0872. CHN analysis: for C9H16O5 calculated
C, 52.93%; H, 7.90%; found: C, 52.84%; H, 7.81%.
(DME, 5 mL) was cooled to 0 ꢁC. A solution of diethyl
cyanomethylphosphonate (550 lL, 3.40 mmol) in DME
was added dropwise, and the stirring was continued for
10 min at 0 ꢁC. A solution of the sugar 10 (760 mg,
1.70 mmol) in DME (5.0 mL) was then added, cooling
was removed, and stirring continued for 4 h. The reac-
tion was quenched with water and extracted with tert-
butyl methyl ether (3·). The combined organic extracts
were washed with brine, dried with anhydrous magne-
sium sulfate, and filtered, and the solvent was evapo-
rated. The crude product (909 mg) was purified by
gradient chromatography (silica-gel, EtOAc/hexanes,
EtOAc: 0–50%). Two epimers were obtained: minor epi-
1
mer (15): 180 mg (23%) H NMR (500 MHz, CDCl3) d:
7.48 (m, 6H), 7.34 (m, 6H), 7.28 (m, 3H), 4.20 (m, 2H),
4.05 (dd, J = 7.8, 5.3 Hz, 1H), 3.33 (d, J = 4.1 Hz, 1H),
2.61 (dd, J = 16.7, 5.3 Hz, 1H), 2.52 (dd, J = 16.7,
8.0 Hz, 1H), 1.55 (s, 3H), 1.38 (s, 3H), 1.37 (s, 3H).
13C NMR (500 MHz, CDCl3) d: 143.58, 128.73,
127.83, 127.12, 116.95, 114.56, 87.61, 87.32, 87.00,
82.28, 80.94, 63.52, 28.11, 26.62, 18.69, 18.39. HRMS
for C30H31NO4 calculated 492.2151 [M+Na]+, found:
492.2149. CHN analysis: for C30H31NO4 calculated C,
76.73%; H, 6.65%; N, 2.98%; found: C, 76.68%; H,
5.9. 10-O-Allyl-20-C-methyl-20,30-O-(1-methylethylidene)-
D-ribofuranoside (13)
A solution of the triol 12 (1.125 g, 5.51 mmol) in DCM
(6 mL) was treated with 2,2-dimethoxypropane (4 mL)
and para-toluenesulfonic acid (187 mg), and stirred at
room temperature for 2 h. The reaction mixture was
then poured onto aqueous solution of sodium bicarbon-
ate (20 mL) and extracted with DCM (3· 50 mL). The
combined extracts were dried, and the solvent was dis-
tilled off at 100 mm Hg pressure at ambient temperature.
The resulting volatile product was used in the next step
1
6.80%; N, 2.81%. Major epimer (16): 390 mg (49%) H
NMR (500 MHz, CDCl3) d: 7.46 (m, 6H), 7.35 (m,
6H), 7.27 (m, 3H), 4.43 (d, J = 0.9 Hz, 1H), 4.25 (t,
J = 4.80 Hz, 1H), 4.03 (dd, J = 7.3, 5.7 Hz, 1H), 3.29
(dd, J = 10.1, 5.3 Hz, 1H), 3.22 (dd, J 10.3, 4.6 Hz,
1H), 2.68 (ABq, J = 16.7, 7.3 Hz, 2H), 1.50 (s, 3H),
1.44 (s, 3H), 1.43 (s, 3H). 13C NMR (500 MHz, CDCl3)
d: 143.37, 128.62, 127.94, 127.21, 117.59, 113.32, 89.21,
89.07, 87.39, 83.56, 81.62, 63.68, 27.73, 27.32, 22.98,
27.32, 22.97, 18.60. HRMS for C30H31NO4 calculated
492.2151 [M+Na]+, found: 492.2139. CHN analysis:
for C30H31NO4 calculated C, 76.73%; H, 6.65%; N,
2.98%; found: C, 76.69%; H, 6.83%; N, 2.97%.
1
as obtained. H NMR (500 MHz, CDCl3) d: 5.90 (m,
1H), 5.30 (dd, J = 15.6, 1.4 Hz, 1H), 5.22 (dd, J = 10.5,
1.4 Hz, 1H), 4.49 (s, 1H), 4.26 (m, 2H), 3.69 (dd,
J = 12.4, 2.5 Hz, 1H), 3.61 (dd, J = 12.4, 3.7 Hz, 1H),
1.46 (br s, 6H), 1.41 (s, 3H). 13C NMR (500 MHz,
CDCl3) d: 133.20, 117.92, 112.45, 108.93, 92.23, 88.18,
87.39, 69.23, 63.89, 28.05, 27.63, 19.76.
5.10. 10-O-Allyl-20-C-methyl-20,30-O-(1-methylethylid-
ene)-50-O-trityl-D-ribofuranoside (14)
5.12. (10S or 10R)-1-[4-amino-5-cyano-1-(ethoxycarbonyl)-
1H-pyrrol-3-yl]-1,4-anhydro-20-C-methyl-2,3-O-(1-meth-
ylethylidene)-50-O-trityl-D-ribitol (20, 21)
A solution of alcohol 13 (1.77 g, max. 5.51 mmol) and
trityl chloride (1.536 g, 5.51 mmol) in pyridine (6 mL)
was stirred at 60 ꢁC overnight. The solvent was evapo-
rated and the residue was purified by gradient chroma-
tography (silica-gel, EtOAc/hexanes EtOAc: 0–30%) to
yield 1.4554 g (54%, two steps) of the desired product.
1H NMR (500 MHz, CDCl3) d: 7.48 (m, 6H), 7.30 (m,
9H), 5.65 (m, 1H), 5.08(m, 2H), 4.34 (dd, J = 8.5,
5.5 Hz, 1H), 4.00 (dd, J = 13.3, 4.3 Hz, 1H), 3.84 (dd,
J = 13.3, 5.5 Hz, 1H), 3.28 (dd, J = 9.4, 5.5 Hz, 1H),
3.10 (t, J = 9.4 Hz, 1H), 1.51 (s, 3H), 1.45 (s, 3H), 1.28
(s, 3H). 13C NMR (500 MHz, CDCl3) d: 143.81,
128.64, 127.77, 126.99, 116.25, 112.45, 108.53, 91.42,
88.37, 86.68, 84.66, 67.94, 64.06, 28.10, 27.71. HRMS
for C31H34O5 calculated 509.2304 [M+Na]+, found:
509.2290. CHN analysis: for C31H34O5 calculated C,
76.52%; H, 7.04%; found: C, 76.49%; H, 7.29%.
A solution of diisopropyl amine (1.42 mL, 10 mmol) in
THF (40 mL) was cooled to ꢀ78 ꢁC and treated drop-
wise with n-butyllithium (4.00 mL, 10 mmol, 2.5 M solu-
tion in hexanes). To this mixture, a solution of 16
(2.22 g, 4.78 mmol) in THF (60 mL) was added at
ꢀ78 ꢁC, via syringe. Stirring at cold was continued for
90 min, after which time neat methyl formate
(1.00 mL, 16.20 mmol) was added. The reaction mixture
was stirred at ꢀ78 ꢁC for another 2 h, and the dry ice
cooling bath was replaced with an ice bath. After
20 min the reaction was quenched by pouring onto a
10% aqueous solution of citric acid. The product was ex-
tracted with CHCl3 (4· 100 mL), the combined extracts
were washed with brine (1· 100 mL) and dried (anhy-
drous sodium sulfate), and the solvent was evaporated.
The crude 17 (2.67 g) was dissolved in MeOH (20 mL),
and water was added (2.0 mL) followed by sodium ace-
tate (540 mg, 6.60 mmol) and aminoacetonitrile hydro-
chloride (610 mg, 6.60 mmol). The reaction mixture
was stirred at ambient temperature for 72 h. It was di-
luted with CHCl3 and washed with water. The aqueous
5.11. 20-C-Methyl-20,30-O-(1-methylethylidene)-50-O-tri-
tyl-a-D-ribofuranos-10(S or R)-yl-acetonitrile (15, 16)
A stirred suspension of sodium hydride (120 mg,
3.0 mmol, 60% in mineral oil) in dimethoxyethane

5226
G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
phase was back-washed with CHCl3, the combined or-
ganic phase was dried, and the solvent was evaporated.
The crude product (18, 2.44 g) was dissolved in DCM
(20 mL) and DBU (1.34 g, 8.80 mmol) was added. To
this mixture ethyl chloroformate (716 lL, 6.60 mmol)
was added via syringe, and stirring at room temperature
was continued for 2 h. At this time, LC–MS indicated
full formation of the urethane 19, and additional DBU
(1.34 g, 8.80 mmol) was added to induce the cyclization.
The stirring at room temperature was continued over-
night. The reaction mixture was diluted with CHCl3
(50 mL) and extracted with an aqueous solution of citric
acid (10%, 2· 50 mL). The combined aqueous phases
were washed with CHCl3, the combined organic extracts
were dried (anhydrous sodium sulfate), and the solvent
was evaporated. The crude product (2.38 g) was purified
by column chromatography (silica-gel, dichlorometh-
ane/diethyl ether 98:2, isocratic) to obtain two epimers:
1 mg/mL, CDCl3): irradiation of the singlet at 5.22
ppm (10-H) resulted in a pronounced enhancement of
the multiplet at 4.29 ppm (40-H) by 4%. 13C NMR
(500 MHz, CDCl3) d: 143.68, 141.72, 128.73, 127.83,
127.13, 120.84, 114.94, 114.51, 109.47, 89.54, 87.04,
86.89, 86.43, 82.59, 82.41, 63.71, 31.57, 28.21, 26.69,
22.63, 19.80, 14.10. HRMS for C33H33N3O4 calculated
558.2369 [M+Na]+, found: 558.2374. CHN analysis:
for C33H33N3O4 calculated C, 74.00%; H, 6.21%; N,
7.84%; found: C, 73.75%; H, 6.93%; N, 7.06%.
5.14. (10S)-1-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-
1,4-anhydro-2-C-methyl-20,30-O-(1-methyl-ethylidene)-50-
O-trityl-D-ribitol (23)
A solution of the nitrile 22 (182 mg, 0.339 mmol) and
formamidine acetate (354 mg, 3.40 mmol) in EtOH
(6 mL) was heated with stirring in a sealed tube to
90 ꢁC for 5 h. After cooling to ambient temperature,
the solvent was evaporated. Water was added, and the
product was extracted with tert-butyl methyl ether
(4·). The combined organic extracts were back washed
with brine and dried, and the solvent was evaporated
to yield 173 mg of crude product. It was further purified
by preparative TLC (DCM + MeOH/9:1) to obtain
1
minor epimer (20, higher Rf, 280 mg, 12%): H NMR
(500 MHz, CDCl3) d: 7.52, (m, 6H), 7.36 (m, 6H), 7.30
(m, 3H), 4.76 (s, 1H), 4.57 (br s, 2H), 4.44 (q,
J = 7.1 Hz, 2H), 4.40 (d, J = 2.50 Hz, 1H), 4.32 (dd,
J = 6.7, 3.9 Hz, 1H), 3.43 (dd, J = 10.5, 3.5 Hz, 1H),
3.36 (dd, J = 10.5, 4.5 Hz, 1H), 1.63 (s, 3H), 1.47 (t,
J = 7.1 Hz, 2H), 1.43 (s, 3H), 1.24 (s, 3H). 13C NMR
(500 MHz, CDCl3) d: 146.11, 143.57, 128.72, 127.81,
127.15, 122.20, 114.69, 89.15, 86.95, 86.90, 82.68,
81.68, 64.18, 63.78, 31.54, 28.08, 26.50, 22.60, 19.62,
14.10, 14.07. HRMS for C36H37N3O6 calculated
630.2580 [M+Na]+, found: 630.2595. CHN analysis:
for C36H37N3O6 calculated C, 71.15%; H, 6.14%; N,
6.91%; found: C, 71.48%; H, 6.45%; N, 6.59%. Major
epimer (21, lower Rf, 1.25 g, 56%): 1H NMR
(500 MHz, CDCl3) d: 7.48 (m, 6H), 7.34 (m, 6H), 7.26
(m, 3H), 5.31 (s, 1H), 4.71 (s, 1H), 4.47 (m, 3H), 4.33
(t, J = 5.0 Hz, 1H), 3.33 (dd, J = 10.3, 5.3 Hz, 1H),
3.27 (dd, J = 10.3, 5.0 Hz, 1H), 1.57 (s, 3H), 1.45 (m,
9H). 13C NMR (500 MHz, CDCl3) d: 143.39, 128.61,
127.94, 127.25, 123.55, 123.00, 90.40, 88.07, 87.35,
83.19, 82.59, 64.29, 63.37, 27.63, 27.53, 14.08. HRMS
for C36H37N3O6 calculated 630.2580 [M+Na]+, found:
630.2570. CHN analysis: for C36H37N3O6 calculated
C, 71.15%; H, 6.14%; N, 6.91%; found: C, 71.20%; H,
6.11%; N, 6.65%.
1
137 mg of pure product. H NMR (500 MHz, CD3OD)
d: 8.16 (s, 1H), 7.50 (m, 6H), 7.32 (m, 6H), 7.26 (m, 4H),
5.29 (s, 1H), 4.30 (d, J = 3.0 Hz, 1H), 4.23 (m, 1H), 3.40
(dd, J = 5.0, 1.6 Hz, 2H), 1.64 (s, 3H), 1.38 (s, 3H), 1.15
(s, 3H). 13C NMR (500 MHz, CD3OD) d: 149.92,
145.22, 129.95, 129.90, 129.19, 128.88, 128.25, 115.84,
112.41, 90.66, 89.07, 88.26, 83.31, 83.22, 65.27, 28.59,
27.29, 21.77. HRMS for C34H34N4O4 calculated
563.2658 [M+H]+, found: 563.2662. CHN analysis: for
C34H34N4O5 (0.5 H2O) calculated C, 71.43%; H,
6.17%; N, 9.80%; found: C, 71.51%; H, 6.00%; N,
8.52%.
5.15. (10S)-1-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-
1,4-anhydro-20-C-methyl-D-ribitol (3)
A solution of the protected nucleoside 23 (105 mg,
0.1966 mmol) was dissolved in MeOH (2.0 mL, anhy-
drous) and treated with a solution of hydrogen chloride
in dioxane (4 N, 200 lL). Stirring at ambient tempera-
ture was continued for 24 h, after which the solvent
was evaporated. Water was added, and the product
was extracted with tert-butyl methyl ether (3·). The
aqueous phase was heated briefly to reflux with char-
coal, micro-filtered, and the solvent was evaporated to
dryness. The residue was triturated with 2 mL of aceto-
nitrile. The solid was washed two more times with cold
acetonitrile and dried on high-vacuum to afford 33.7 mg
(61%) of the desired product. 1H NMR (600 MHz,
CD3OD) d: 8.36 (s, 1H, 2-H), 7.73 (s, 1H, 8-H), 5.11
(s, 1H, 10-H), 4.05 (dd, J = 11.8, 2.5 Hz, 1H, 500-H),
3.97 (dt, J = 7.7, 2.5 Hz, 1H, 40-H), 3.93 (dd, J = 11.7,
2.6 Hz, 50-H), 3.82 (d, J = 7.6 Hz, 1H, 30-H), 0.92 (s,
5.13. (10S)-1-(4-Amino-5-cyano-1H-pyrrol-3-yl)-1,4-anhy-
dro-20-C-methyl-20,30-O-(1-methylethylidene)-5-O-trityl-
D-ribitol (22)
A solution of the urethane 20 (207 mg, 0.341 mmol) in
EtOH (6 mL) was treated with potassium carbonate
(100 mg) and stirred at ambient temperature for 1 h.
The reaction was quenched with water and extracted
with CHCl3/iPrOH (85:15, 3·). The combined organic
extracts were dried with anhydrous sodium sulfate and
the solvent was evaporated. The crude product
(182 mg, 100%) was used in the next step without addi-
1
tional purification. H NMR (500 MHz, CDCl3) d: 8.0
(s, 1H), 7.56 (m, 6H), 7.33 (m, 6H), 7.27 (m, 3H), 6.68
(d, J = 2.1 Hz, 1 H), 4.82 (s, 1H), 4.33 (d, J = 2.80
1H), 4.29 (m, 1H), 4.12 (s, 2H), 3.42 (dd, J = 10.3,
3.9 Hz, 1H), 3.35 (dd, J = 10.3, 4.6 Hz, 1H), 1.63
(s, 3H), 1.41 (s, 3H), 1.21 (s, 3H). nOe (600 MHz,
3H, C0 -CH3). 13C NMR (600 MHz, CD3OD) d: 154.50
2
(C6), 145.91 (C8), 133.59, 130.49 (C2), 84.66 (C01), 83.87
(C40 ), 79.74 (C20 ), 75.36 (C30 ), 61.16 (C05), 22.60
(C02-CH3). HRMS for C12H16N4O5 calculated 281.1250
[M+Na]+, found: 281.1265.

G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
5227
5.16. (10R)-1-(4-amino-5-cyano-1H-pyrrol-3-yl)-1,4-anhydro-
20-C-methyl-20,30-O-(1-methylethylidene)-5-O-trityl-D-ribitol
(24)
hydrogen chloride in dioxane (4 N, 200 lL) and stirred
at ambient temperature for 24 h. The solvent was evap-
orated and the residue was dissolved in water. After
extraction with DCM (3·) the combined organic ex-
tracts were washed with water (1·). The combined
aqueous phase was decolorized with charcoal and
evaporated to dryness. The crude product (98 mg)
was recrystallized from acetonitrile to yield 56 mg
(77%) of the pure product. 1H NMR (500 MHz,
CD3OD) d: 8.36 (s, 1H, 2-H), 7.75 (s, 1H, 8-H), 4.95
(s, 1H, 10-H), 4.17 m (1H, 40-H), 4.06 (d, J = 8.0 Hz,
1H, 30-H), 3.86 (dd, J = 11.9, 1.6 Hz, 1H, 500-H), 3.70
(dd, J = 11.9, 4.4 Hz, 1H, 50-H), 1.19 (s, 3H,
A solution of the urethane 11 (1.076 g, 1.77 mmol) in
EtOH (20 mL) was treated with potassium carbonate
(500 mg) and stirred at ambient temperature for 1 h.
Water was added and the mixture was extracted with
CHCl3/iPrOH (85:15, 3· 50 mL). The combined organic
extracts were dried with anhydrous sodium sulfate, and
the solvent was evaporated. The crude product (995 mg,
ꢁ100%) was purified by gradient chromatography (sil-
ica-gel, EtOAc/hexanes, EtOAc: 0–100%) to obtain
704 mg (74%) of pure product. 1H NMR (500 MHz,
CDCl3) d: 8.36 (s, 1H), 7.48 (d, J = 7.3 Hz, 6H), 7.34 (t,
J = 7.3 Hz, 6H), 7.28 (t, J = 7.1 Hz, 3H), 6.76 (d,
J = 3.44 Hz, 1H), 4.77 (s, 1H), 4.51 (d, J = 1.1 Hz, 1H),
4.29 (t, J = 4.8 Hz, 1H), 3.36 (dd, J = 10.1, 5.3 Hz, 1H),
3.30 (dd, J = 10.1, 5.0 Hz, 1H), 2.00 (s, 2H), 1.54 (s,
3H), 1.46 (s, 3H), 1.44 (s, 3H). nOe (2 mg/mL, CDCl3):
irradiation of the singlet at 4.78 ppm (10-H) resulted in a
weak enhancement of the doublet at 4.48 ppm (30-H) by
<1%. 13C NMR (600 MHz, CD3OD) d: 143.49, 128.59,
127.88, 127.15, 123.09, 114.92, 112.86, 107.61, 90.49,
88.29, 87.19, 87.10, 82.79, 81.88, 63.71, 28.11, 27.79,
22.27. HRMS for C33H33N3O4 calculated 558.2369
[M+Na]+, found: 558.2350. CHN analysis: for
C33H33N3O4 calculated C, 74.00%; H, 6.21%; N, 7.84%;
found: C, 73.73%; H, 5.93%; N, 7.70%.
C0 -CH3). 13C NMR (600 MHz, CD3OD) d: 154.50
2
(C6), 145.90 (C7), 135.32, 132.11 (C2), 114.88, 109.97,
83.39 ( C04), 82.13 (C10 ), 79.66 (C20 ), 77.46 ( C03), 63.34
(C05), 20.31 (C20 -CH3). HRMS for C12H16N4O5 calcu-
lated 281.1250 [M+Na]+, found: 281.1249.
5.19. (10S or 10R)-1-(4-Amino-5-cyano-3-furyl)-1,4-anhy-
dro-20-C-methyl-20,30-O-(1- methylethylidene)-5-O-trityl-
D-ribitol (28, 29)
The solution of the hydroxymethylene derivative 17
(260 mg, max. 0.50 mmol) in DMF (4 mL) was treated
with chloroacetonitrile (300 lL, large excess), cesium
carbonate (700 mg) was added and stirred at ambient
temperature overnight. The reaction mixture was
poured into water (20 mL) and extracted with tert-butyl
methyl ether (3· 30 mL). The combined organic phase
was washed with brine and dried (anhydrous sodium
sulfate) and the solvent was evaporated to dryness.
The crude 27 (222 mg) was dissolved in THF (4 mL)
and added drop-wise into a solution of LDA [prepared
from nBuli (2.5 M in hexanes, 2.1 mL, 5.20 mmol) and
diisopropylamine (730 lL, 5.20 mmol)] at ꢀ78 ꢁC via
syringe. The reaction mixture was stirred at ꢀ78 ꢁC
for 2 h and was quenched with a saturated solution of
ammonium chloride (20 mL). The crude product was ex-
tracted with tert-butyl methyl ether (4· 30 mL), the
combined organic extract was washed with brine
(1· 30 mL) and dried with anhydrous magnesium sul-
fate, and the solvent was evaporated. The crude product
was purified by gradient chromatography (silica-gel,
EtOAc/hexanes, EtOAc: 0–70%) to obtain 17.2 mg
(3%) of the b-epimer 28 and 73 mg (9%) of a-epimer
5.17. (1R)-1-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-
1,4-anhydro-2-C-methyl-2,3-O-(1-methyl ethylidene)-5-
O-trityl-D-ribitol (25)
A solution of the nitrile 24 (700 mg, 1.307 mmol) and
formamidine acetate (1.36 g, 13.07 mmol) in EtOH
(10 mL) was heated with stirring in a sealed tube to
90 ꢁC for 3 h. The solvent was evaporated and water
(30 mL) was added. The product was extracted with
tert-butyl methyl ether (4· 250 mL), the combined extract
was washed with brine (1· 50 mL), dried (anhydrous so-
dium sulfate), and the solvent was evaporated. The crude
product (705 mg) was purified by gradient chromatogra-
phy (silica-gel, MeOH/DCM, MeOH: 0–15%) to obtain
607 mg (82%) of pure product. 1H NMR (500 MHz,
CD3CN) d: 9.62 (s, 1H), 8.21 (s, 1H), 7.50 (m, 6H), 7.33
(m, 6H), 7.26 (m, 3H), 5.68 (s, 1H), 5.28 (s, 1), 4.36 (d,
J = 0.7 Hz, 1H), 4.18 (t, J = 5.72 Hz, 1H), 3.34 m (dd,
J = 10.1, 6.4 Hz, 1H), 3.20 (dd, J = 10.7, 5.5, 1H), 1.49
(s, 3H), 1.34 (s, 3H), 1.33 (s, 3H). 13C NMR (500 MHz,
CD3CN) d: 151.61, 151.08, 147.75, 144.93, 129.75,
129.60, 128.93, 128.15, 118.29, 114.17, 113.18, 112.11,
90.84, 89.76, 87.97, 83.66, 80.35, 63.98, 28.44, 28.08,
23.08. HRMS for C34H34N4O4 calculated 563.2658
[M+H]+, found: 563.2662. CHN analysis: for
C34H34N4O4 calculated C, 72.58%; H, 6.09%; N, 9.96%;
found: C, 72.70%; H, 6.16%; N, 9.72%.
1
29. Minor epimer (28): H NMR (500 MHz, CDCl3) d:
7.48 (m, 6H), 7.34 (m, 6H), 7.28 (m, 3H), 7.22 (s, 1H),
4.74 (s, 1H), 4.31 (m,3H), 3.41 (dd, J = 10.3, 3.9 Hz,
1H), 3.36 (dd, J = 10.5, 4.6 Hz, 1H), 1.62 (s, 3H), 1.41
(s, 3H), 1.24 (s, 3H). 13C NMR (600 MHz, CDCl3) d:
143.61, 143054, 143.44, 128.70, 127.85, 127.20, 115.43,
114.76, 112.81, 89.00, 87.00, 86.91, 82.77, 81.00, 63.54,
29.67, 28.11, 26.53, 19.55. HRMS for C33H32N2O5 cal-
culated 559.2203 [M+Na]+, found: 559.2240. Major epi-
mer (29): 1H NMR (500 MHz, CD3OD) d: 7.48 (m, 6H),
7.35 (m, 6H), 7.30 (m, 4H), 4.74 (s, 1H), 4.52 (d,
J = 0.7 Hz, 1H), 4.35 (m, 3H), 3.38 (dd, J = 10.3,
5.0 Hz, 1H), 3.30 (dd, J = 10.1, 5.0 Hz, 1H), 1.52 (s,
3H), 1.47 (s, 3H), 1.46 (s, 3H). 13C NMR (600 MHz,
CD3OD) d: 145.62, 144.69, 143.36, 128.67, 128.58,
127.92, 127.82, 127.24, 127.17, 113.84, 113.13, 112.76,
110.41, 90.34, 88.12, 87.33, 83.35, 80.87, 63.48, 31.52,
5.18. (10R)-1-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-
1,4-anhydro-20-C-methyl-D-ribitol (26)
A solution of 25 (148 mg, 0.263 mmol) in MeOH
(2.0 mL, anhydrous) was treated with a solution of

5228
G. Butora et al. / Bioorg. Med. Chem. 15 (2007) 5219–5229
27.83, 27.56, 22.40. HRMS for C33H32N2O5 calculated
559.2203 [M+Na]+, found: 559.2186.
C33H32N2O5 calculated 559.2203 [M+Na]+, found:
559.2246.
5.20. (10S)-1-(4-aminofuro[3,2-d]pyrimidin-7-yl)-1,4-anhy-
dro-20-C-methyl-20,30-O-(1-methylethylidene)-5-O-trityl-D-
ribitol (30)
5.23. (10R)-1-(4-Aminofuro[3,2-d]pyrimidin-7-yl)-1,4-anhy-
dro-20-C-methyl-D-ribitol (32)
A solution of the protected nucleoside 31 (23 mg,
0.041 mmol) was dissolved in MeOH (3.0 mL), treated
with 1.0 mL of 4 N solution of HCl in dioxane, and stir-
red at ambient temperature for 24 h. The reaction mix-
ture was distributed between water (10 mL) and DCM
(20 mL), and the aqueous phase was extracted with
DCM (2·). The combined organic extract was washed
with water (6 mL) and the combined aqueous phase
was decolorized with charcoal and filtered through Cel-
ite. The filtrate was concentrated to a volume of about
5 mL, micro-filtered, and the remaining solvent was
evaporated to dryness. The residue was recrystallized
from acetonitrile to afford 8.4 mg (73%) of the desired
nucleoside. 1H NMR (500 MHz, CD3OD) d: 8.49 (s,
1H), 8.32 (s, 1H), 4.96 (s, 1H), 4.19 (br s, 1H), 4.04 (d,
J = 7.8 Hz, 1H), 3.84 (d, J = 9.6 Hz, 1H), 3.68 (dd,
J = 11.9, 3.4 Hz, 1H), 1.22 (s, 3H). 13C NMR
(500 MHz, CD3OD) d: 151.43, 148.29, 83.79, 80.14,
77.27, 63.17, 40.05, 20.27. 13C NMR (500 MHz,
CD3OD) d: 153.27, 150.19, 149.18, 118.68, 84.55,
82.37, 79.56, 75.40, 22.34. HRMS for C12H15N3O5 cal-
culated 281.1012, found: 282.1018. [M+H]+.
A solution of the amino nitrile 28 (37 mg, 0.069 mmol)
in EtOH (3 mL) was treated with formamidine acetate
(214 mg, 2.07 mmol) and heated to 85 ꢁC in a sealed
tube for 12 h. The solvent was evaporated, and the crude
product
was
purified
by
preparative
TLC
(DCM + MeOH/9:1) to obtain 14.0 mg (36%) of the de-
1
sired product. H NMR (500 MHz, CD3OD) d: 8.24 (s,
1H), 7.88 (s, 1H), 7.50 (m, 6H), 7.31 (m, 6H), 7.25 (m,
3H), 5.17 (s, 1H), 4.30 (d, J = 3.0 Hz, 1H), 4.25 (m,
1H), 3.34 (d, J = 5.04 Hz, 2H), 1.63 (s, 3H), 1.37 (s,
3H), 1.17 (s, 3H). 13C NMR (500 MHz, CD3OD) d:
154.42, 148.51, 145.23, 129.95, 128.87, 128.25, 120.04,
115.85, 90.70, 89.18, 88.23, 83.78, 82.65, 65.21, 28.64,
27.31, 21.69. HRMS for C34H33N3O5 calculated
586.2312 [M+Na]+, found: 586.2302.
5.21. (10S)-1-(4-Aminofuro[3,2-d]pyrimidin-7-yl)-1,4-anhy-
dro-2-C-methyl-D-ribitol (4)
A solution of the protected nucleoside 30 (15.7 mg,
0.028 mmol) in MeOH (3 mL) was treated with a solu-
tion of HCl in dioxane (4 N, 1 mL) and stirred at ambi-
ent temperature for 2 h. The solvent was evaporated,
and the residue was partitioned between water (10 mL)
and DCM (20 mL). The aqueous phase was extracted
with DCM (2·), treated with charcoal, and filtered
through Celite. The filtrate was concentrated to a vol-
ume of about 5 mL, micro-filtered, and evaporated to
dryness. Trituration with acetonitrile afforded 7.0 mg
(88%) of the clean nucleoside. 1H NMR (500 MHz,
CD3OD) d: 8.47 (s, 1H), 8.26 (s, 1H), 5.09 (s, 1H),
4.02 (d, J = 13.0 Hz, 1H), 3.96 (br s, 1H), 3.87 (d,
J = 11.7 Hz, 1H), 3.82 (d, J = 7.4 Hz, 1H), 1.00 (s,
3H). 13C NMR (600 MHz, CD3OD) d: 153.27, 150.19,
149.18, 118.68, 84.55, 82.36, 79.56, 75.40, 61.43, 22.33.
HRMS for C12H15N3O5 calculated 281.1012, found:
282.1044. [M+H]+.
References and notes
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