Synthesis and biological evaluation of 5R- and 5S-methyl substituted D- and L-configuration 1,3-dioxolane nucleoside analogs

Article abstract of DOI:10.1016/j.bmc.2004.08.054

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-meth

Full text of DOI:10.1016/j.bmc.2004.08.054

Bioorganic & Medicinal Chemistry 12 (2004) 6237–6247
Synthesis and biological evaluation of 5R- and
5S-methyl substituted D- and L-configuration 1,3-dioxolane
nucleoside analogs
Sanjib Bera,a Leila Malik,a Balkrishen Bhat,a Steven S. Carroll,b Renee Hrin,b
Malcolm MacCoss,c Daniel R. McMasters,c Michael D. Miller,b Greg Moyer,b
David B. Olsen,b William A. Schleif,b Joanne E. Tomassinib and Anne B. Eldrupa,*
aDepartment of Medicinal Chemistry, Isis Pharmaceuticals, Carlsbad, CA 92008, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 2 June 2004; revised 31 August 2004; accepted 31 August 2004
Abstract—1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemother-
apy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxy-
methyl)-5-methyldioxolane. Dioxolanes of a-D-, b-D-, a-L-, and b-L-configuration were prepared, that included 5-methyl
derivatives of both 5R and 5S configuration. Molecular mechanics calculations indicate that the 5S and 5R diastereoisomeric
1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference
of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic repl-
icon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-
dioxolanes were found to be inactive.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1.Introduction
such as oxygen or sulfur in place of the 30-methylene car-
bon. The best known example of this class of com-
pounds is Lamivudine also known as 3TC or (ꢀ)-L-b-
1,3-oxathiolanyl cytosine.1–8 The 1,3-dioxolane and
1,3-oxathiolane nucleoside analogs display broad anti-
viral and anti-neoplastic activities (Fig. 1). Lamivudine,
1, is efficacious against immunodeficiency virus (HIV)
and hepatitis B virus (HBV) infections. The correspond-
ing oxa derivative, (ꢀ)-L-b-1,3-dioxanyl cytosine, 2, pos-
sesses anti-neoplastic activity,9 in addition to its activity
against HIV and HBV.5 In the purine series, (ꢀ)-D-b-
1,3-dioxanylguanine (DGX), 3, and (ꢀ)-D-b-1,3-dioxa-
nyl-2,6-diaminopurine (DAPG), 410 (Fig. 1), are under-
going pre-clinical evaluation as anti-HIV11 and anti-
HBV agents.11,12 In addition, (+)-L-b-1,3-oxathionyl
adenine, 5,13 has demonstrated anti-HIV activity in
PBMCs. Furthermore, dioxolane based nucleoside tri-
phosphates have been reported to be inhibitors of
RNA synthesis mediated by the hepatitis C virus
RNA-dependent RNA polymerase (HCV RdRp).14,15
Nucleoside analogs play a central role in anti-viral and
anti-neoplastic chemotherapy. Examples of nucleoside
analogs approved by the FDA for the treatment of viral
conditions include Zidovudine (AZT, Retrovirꢁ),
Didanosine (ddI, Videxꢁ), Zalcitabine (ddC, Hividꢁ),
Lamivudine (3TC, Epivirꢁ), and Stavudine (4dT, Zer-
itꢁ), that are approved for the treatment of HIV infec-
tion. In addition, Fludarabine (Fludaraꢁ), Cladribine
(2-CdA, Leustatinꢁ), and Pentostatin (DCF, Nipentꢁ)
are examples of nucleoside analogs approved for the
treatment of neoplastic conditions. Subsequent to the
successful introduction of AZT for the treatment of
HIV infection, significant effort has been put toward
the discovery and development of more efficacious and
selective nucleoside analogs. A particularly successful
class of nucleoside analogs incorporates a heteroatom
Keywords: Dioxolanes; Nucleoside anti-virals; HCV; HIV; Vaccinia.
*
We recently described the methyl substituted nucleosides
20-C-methyladenosine and 20-C-methylguanosine as
Corresponding author. Tel.: +1 760 603 3852; fax: +1 760 603
4654; e-mail: aeldrup@isisph.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.054

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S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
Figure 1. Examples of 1,3-dioxolane and 1,3-oxathiolane nucleosides that display anti-viral or anti-neoplastic activities: (1) Lamivudine, 3TC, (ꢀ)-b-
L-(2R,5S)-oxathiolanyl-cytosine; (2) O-ddC, (ꢀ)-b-L-(2S,4S)-dioxolanyl-cytosine; (3) DXG, (ꢀ)-b-D-(2R,4R)-dioxolanyl-guanine; (4) DAPD, (ꢀ)-b-
D-(2R,4R)-dioxolanyl-2,6-diaminopurine; (5) (+)-b-L-(2R,5S)-oxathiolanyl-adenine.
inhibitors of HCV RNA replication in cells.16–18 The tri-
Table 1.
phosphates of these nucleosides were found to be com-
petitive inhibitors of the HCV polymerase, suggesting
that the 20-C-methyl modification is tolerated by the
HCV viral polymerase, as well as by the kinases respon-
sible for the metabolism of the 20-C-methylribonucleo-
sides to their triphosphates. Moreover, inclusion of the
methyl substituent in the C20 position was found to pro-
mote a conformational bias toward the C30-endo (north)
ribonucleoside conformer,16 and was found to attenuate
cytotoxicity for a nucleoside that incorporated a modi-
fied purine heterobase (Olsen et al. unpublished results;
Eldrup et al., unpublished results).
HCV replicon
% inhibition
at 100lM
Vaccinia
% inhibition
at 100lM
HIV spread
IC95 (lM)
25a
25b
26a
26b
30a
30b
31a
31b
34
No data
<30
46
<30
<30
54
No data
>10
>10
<30
<30
<30
<30
<30
<30
<30
<30
<30
<30
<30
<30
<30
<30
>10
>10
>10
>10
>10
>10
35
>10
Dioxolane based nucleoside analogs that incorporate
methyl substitutions in positions that correspond to
the ribonucleoside C40 and C30 positions have been re-
ported.19,20 However, dioxolanes with methyl substitu-
ents in what would correspond to the nucleoside C20
position have not been described. Based on the findings
outlined above, we recently reported preliminary work
toward the synthesis of 2S-hydroxymethyl-1,3-dioxo-
lanes (b-D-configuration) and (2S,5R)-2-hydroxy-
of general structure 6, 7, and 8 (Fig. 2) were selected
as synthetic targets to ensure variations in configuration
(D and L) and in the stereochemical positioning (5R/beta
face or 5S/alpha face) of the methyl substituent. A
change in conformational bias might affect biological
activity and specificity and hence, molecular mechanics
calculations were applied to assess the effect of 5-methyl
substitution on conformational preference. Compounds
were evaluated as inhibitors of HCV RNA replication in
a replicon assay in HB-1 cells, as inhibitors of HIV
spread in human T-lymphoid cells and as inhibitors of
vaccinia virus replication (Table 1).
methyl-5-methyl-1,3-dioxolanes
(b-D-configuration)
that carried pyrrolo[2,3-d]pyrimidine heterobases.21 We
now report the synthesis and biological evaluation of
5-methyl substituted L- and D-configuration dioxolane
nucleosides carrying purine heterobases. Compounds
2.Results
2.1. Chemistry
For the precursor for the D-configuration dioxolanes of
general structure 6 (Fig. 2), 2-benzoyloxyacetaldehyde 9
was condensed22 with commercially available (4S,5R)-
methyl-2,2,5-trimethyl-1,3-dioxalane-4-carboxylate 10
in the presence of para-toluenesulfonic acid to give 11
as an inseparable 2R,2S diastereoisomeric mixture in
9:1 ratio (Scheme 1). Saponification of the methyl esters
Figure 2. Target compounds: (2R,4R,5R)-5-methyl-1,3-dioxolanyl-
purinenucleosides (b-D-configuration) 6, (2S,4S,5S)-5-methyl-1,3-dioxo-
lanyl-purinenucleosides (b-L-configuration) 7, and (2R,4R,5S)-5-
methyl-1,3-dioxolanyl-purinenucleosides (b-D-configuration) 8. Nucleo-
base, B, is either adenine, diaminopurine or guanine.

S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
6239
11 with lithium hydroxide in aqueous tetrahydrofuran,
followed by acidification gave a mixture of the carboxy-
lic acids 12 and 13, which were separated by silica gel
column chromatography. The stereochemistry of the
carboxylic acid derivative 12 was confirmed by NOE
(upon which irradiation of H-2 resulted in enhancement
of the H-5 signal, confirming the b-configuration of the
benzyloxymethyl substituent at C-2). Oxidative decarb-
oxylation of the b-configuration (2,4-trans) carboxylic
acid derivative 12 with lead tetraacetate in acetonitrile,
in the presence of pyridine, gave acetyl intermediate 14
as a 4R,4S-diastereoisomeric mixture. Compound 14
served as the starting material for the synthesis of the
D-configuration, 5R-methyl dioxolanes 25a,b and 26a,b
(Scheme 3).
(4R,5S)-methyl-2,2,5-trimethyl-1,3-dioxolane-4-carboxy-
late 15. Condensation of 15 with benzyloxyaldehyde 9
resulted in a 2R,2S diastereoisomeric mixture of the
methylester derivatives 16 in 1:8 ratio (Scheme 2).
Saponification of the methyl ester with aqueous lithium
hydroxide was followed by acidification to afford the
carboxylic acid derivatives 17 and 18, which were sepa-
rable by silica gel column chromatography. The assign-
ment of the stereochemistry of the major isomer of 17
was performed by NOE. Conversion of 17 and 18,
respectively, to their corresponding diastereomeric mix-
tures of the acetoxy derivatives 19 and 20 was achieved
by lead tetraacetate mediated oxidation in acetonitrile,
in the presence of pyridine. Compounds 19 and 20
served as precursors for the L-configuration 5S-methyl
dioxolanes (30a,b and 31a,b, Scheme 4) and the D-con-
figuration 5S-methyl dioxolanes (34 and 35, Scheme
5), respectively.
Using a similar methodology to the one described
above, the precursors for the L-configuration dioxolane
derivatives of general structure 7 and the D-configura-
tion dioxolane derivatives of general structure 8 (Fig.
2) were available from the commercially available
Guanine and 2,6-diaminopurine derivatives of D-configu-
ration dioxolanes were synthesized that contained
Scheme 1. Reagents and conditions: (i) para-toluenesulfonic acid, toluene, 80ꢂC, 1h; (ii) a. 1M aqueous lithium hydroxide/tetrahydrofuran (1:1), b.
1N HCl or sulfuric acid (pH2–3); (iii) lead tetraacetate, acetonitrile, pyridine.
Scheme 2. Reagents and conditions: (i) para-toluenesulfonic acid, toluene, 80ꢂC, 1h; (ii) a. 1M aqueous lithium hydroxide/tetrahydrofuran (1:1), b.
1N HCl or H2SO4 (pH2–3); (iii) lead tetraacetate, acetonitrile, pyridine.

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S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
Scheme 3. Reagents and conditions: (i) a. bis-(trimethylsilyl)acetamide, 1,2-dichloroethane, b. trimethylsilyl triflate, 14, reflux (for 21); (ii) a. 14,
bromotrimethylsilane, dichloromethane, 0ꢂC to rt, b. tris-[2-(2-methoxyethoxy)ethyl]amine, KOH, 22, acetonitrile (for 22); (iii) hydrazine hydrate,
tetrahydrofuran, reflux; (iv) palladium hydroxide/C, H2, methanol; (v) methanolic ammonia, 55ꢂC; (vi) ammonium formate, Pd/C, methanol.
Scheme 4. Reagents and conditions: (i) a. bis-(trimethylsilyl)acetamide, 1,2-dichloroethane, b. 19, trimethylsilyl triflate, reflux (for 21); (ii) a. 19,
bromotrimethylsilane, dichloromethane, 0ꢂC to rt, b. 27, tris-[2-(2-methoxyethoxy)ethyl]amine, KOH, acetonitrile; (iii) hydrazine hydrate,
tetrahydrofuran, reflux; (iv) palladium hydroxide/C, ethanol, cyclohexene, (v) liquid ammonia, 80ꢂC.
methyl substituents of 5R configuration. For the gua-
nine derivatives, silylated
N2-acetyl-O6-diphenyl-
carbamoylguanine (21) was reacted with 14 in the
presence of trimethylsilyl triflate23 to furnish a separable
1:1 mixture of the N-9 ÔglycosylatedÕ, diastereoisomeric
dioxolanes 23a and 24a. Separate deprotection of 23a

S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
6241
O
N
O
O
N
NPh2
O
N
NPh2
O
N
N
N
N
N
N
O
N
NPh2
NHAc
NHAc
+
BnO
BnO
i
O
O
N
O
O
N
H
NHAc
CH3
ii, iii
CH3
ii, iii
21
32
33
O
N
O
N
N
N
N
NH
NH2
NH
NH2
N
HO
HO
O
O
O
O
CH3
CH3
35
34
Scheme 5. Reagents and conditions: (i) a. bis-(trimethylsilyl)acetamide, 1,2-dichloroethane, b. 20, trimethylsilyl triflate, reflux; (ii) hydrazine hydrate,
tetrahydrofuran, reflux; (iii) palladium hydroxide/C, ethanol, cyclohexene.
and 24a with hydrazine hydrate, followed by transfer
hydrogenolysis, produced the guanine derivatives 25a
(2R,4S,5R; a-D) and 26a (2R,4R,5R, b-D), respectively
(Scheme 3). For the synthesis of 2,6-diaminopurine
derivatives, the acetate 14 was treated with bromotri-
methylsilane24 and the thus formed bromide was treated
with 2-amino-6-chloropurine (22) under phase transfer
and 29b in 1:2 ratio. Treatment of this mixture with
ammonia was followed by catalytic hydrogenation to af-
ford the adenine derivatives 30b (2S,4R,5S; a-L) and 31b
(2S,4S,5S; b-L), which were separated by preparative
HPLC to afford the pure diastereoisomers (see
experimental).
conditions25,26
(tris-[2-(2-methoxyethoxy)ethyl]amine
The D-configuration 5S-methyl guanine derivatives 34
and 35 were obtained by treatment of the acetate 20 with
silylated purine derivative 21 under Vorbruggen condi-
tion.23 Thus treatment of 20 with 21 under reflux with
trimethylsilyl triflate in 1,2-dichloroethane produced
an inseparable, 1:1 mixture of the a- and b-nucleoside
derivatives 32 and 33. This mixture was treated with
hydrazine to give the corresponding guanine derivatives.
Removal of the benzyl protection group was performed
by transfer hydrogenolysis to yield a diastereoisomeric
mixture of 34 and 35. Chromatographic separation gave
the desired 34 (2R,4S,5S; a-D) and 35 (2R,4R,5S; b-D).
and KOH in acetonitrile) to give an inseparable mixture
of 23b and 24b (1.2:1). Treatment of this mixture with
methanolic ammonia at elevated temperature produced
the corresponding 2,6-diaminopurine derivatives. The
benzyl protection group was subsequently removed by
transfer hydrogenolysis using Pd/C and ammonium for-
mate in refluxing methanol to give the diastereoisomeric
25b (2R,4S,5R; a-D) and 26b (2R,4R,5R; b-D), which
were separated by preparative HPLC (see experimental).
Adenine and guanine L-configuration dioxolanes were
synthesized that contained methyl substituents of 5S
configuration. The synthesis of the guanine derivative
commenced from the acetate derivative 19, which upon
treatment with a silylated diphenylcarbamoyl purine
derivative (21) in the presence of trimethylsilyl triflate
gave a 1:1 mixture of 28a and 29a (Scheme 4). Chroma-
tographic separation and removal of the heterobase pro-
tection groups with hydrazine gave the a and b
configuration guanine derivatives. Removal of the ben-
zyl protection group was subsequently performed by
transfer hydrogenolysis to give 30a (2S,4R,5S; a-L)
and 31a (2S,4S,5S; b-L), respectively. For the adenine
derivatives, treatment of 19 with bromotrimethylsilane
gave the bromo derivative, which upon reaction with
6-chloropurine 27, under phase transfer conditions, gave
an inseparable mixture of the dioxolane derivatives 28b
The structure of the 1,3-dioxolane derivatives were
determined by proton NMR and high resolution mass
spectroscopy. The assignment of the ÔanomericÕ configu-
ration was performed on the basis of the previously re-
ported characteristics of the proton NMR
spectra.8,13,27 The chemical shifts of anomeric proton
(H-40) of the 2,4-cis derivatives (based on the orientation
of 20-hydroxymethylene and the 40-heterobase) appeared
downfield relative to the H-40 of the trans isomers. Fur-
thermore, the 20-proton of cis derivatives appeared up-
field from that observed for trans isomers, and the
hydroxymethylene protons of cis derivatives appeared
downfield from those observed for trans derivatives.
These shifts were attributed to the fact that the protons
at the syn-position relative to the purine base are more

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S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
Figure 3. Relative energies in kcal/mol of canonical conformations of 3, 26a, and 35 calculated by molecular mechanics. Global minima of 26a and
35 calculated by molecular mechanics. Methyl substitution of the 1,3-dioxolanes induce different conformational preferences in response to 5R- or
5S-methyl substitution.
deshielded than those in an anti-position relative to the
base.
predicted distinct conformational preferences for 26a
and 35, suggesting that the stereochemical placement
of the methyl substituent is decisive for conformational
preference and that 5-substitution can be applied to alter
conformational preference relative to the otherwise con-
formationally unbiased dioxolane, 3.
2.2. Molecular mechanics calculations
In order to assess the effect of the stereochemistry of the
5-methyl substitution on conformational preference,
molecular mechanics calculations were performed to
determine the relative energies of northern- and south-
ern-type conformers of 26a and 35, which incorporate
methyl substituents of 5R and 5S configuration, respec-
tively (Fig. 3 and Methods). While the unsubstituted
dioxolane 3 is calculated to have nearly equal preference
for northern and southern conformation, introduction
of the methyl substituent causes a modest preference
for northern pucker in the case of 26a and southern in
the case of 35.
The 1,3-dioxolanes were evaluated in vitro as inhibitors
of HCV RNA replication, against HIV spread in human
T-lymphoid cells and against vaccinia. Viral targets were
chosen that encode polymerases that might be expected
to display differences in substrate specificity: HCV
encodes an RNA-dependent RNA polymerase, HIV
encode an RNA- or DNA-dependent DNA polymerase
(reverse transcriptase), whereas vaccinia encodes a
DNA-dependent DNA polymerase as well as a DNA-
dependent RNA polymerase. Despite the fact that both
unsubstituted 1,3-dioxolanes and 1,3-oxathiolanes dis-
play biological activities, none of the methyl substituted
derivatives displayed significant inhibitory activity in
any of the assays tested. This inactivity may be due to
inability on part of these polymerases to accept the
methyl substituted derivatives as substrates. Alterna-
tively, mechanisms of cellular uptake and/or nucleoside
kinases may be unwilling to accept the methyl deriva-
tives as substrates. Based on the fact that 1,3-dioxolanes
were tested that displayed variance in conformational
preference, it seems less likely that unsuitable conform-
ational bias is the underlying explanation for the ob-
served inactivity.
2.3. Biological evaluation
Compounds were evaluated as inhibitors of HCV repli-
cation in a cell-based replicon assay,17 against HIV
spread in human T-lymphoid cells (see Methods) and
against vaccinia in HeLa cells (see Methods).28 None
of the described compounds displayed significant activ-
ity in these assays.
3.Discussion
1,3-Dioxolanes and 1,3-oxathiolanes play a central role
in anti-viral and anti-neoplastic therapies. In light of
their importance, we decided to synthesize and evaluate
their 5-methyl derivatives. A versatile strategy for the
synthesis of L- as well as D-configuration 1,3-dioxolanes
that incorporate methyl substituents of both 5R and 5S
configuration was devised. It was anticipated that the
inclusion of a 5-methyl substituent might impose a con-
formational preference distinct from that of the unsub-
stituted derivatives. If so, such a conformational
preference could contribute to improved affinity and
selectivity for the target polymerases. In order to assess
the effect of 5-methyl substitution on conformational
preference, molecular mechanics calculations were per-
formed on the guanine derivatives 3, 26a, and 35, respec-
tively (Fig. 3 and experimental section). Our calculations
4.Methods
4.1. General methods
Diphenylcarbamic acid 2-acetylamino-9H-purin-6-yl es-
ter 21 was prepared as described. TLC was performed
on silica 60 (Merck 5554 aluminum sheet), column chro-
matograpy on silica 60 (230–400mesh ASTM) (Merck
9385). 1H and 13C NMR spectra were obtained at
200MHz (Varian Mecury VX) in 5mm tubes unless oth-
erwise indicated; chemical shifts are positive in the low-
field direction. FAB mass spectra were recorded on a
Jeol Hx110/110 mass spectrometer.

S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
6243
4.2. Molecular mechanics calculations
zyloxyacetaldehyde 9 (4.53g, 30.1mmol) in toluene
(60mL) was added para-toluenesulfonic acid (0.25g,
1.33mmol). The reaction mixture was heated at 80ꢂC
under vacuum for 1h. The reaction mixture was cooled
down to room temperature and evaporated to dryness.
The residue was purified over silica gel (ethyl acetate/
hexane 1:3) to give the ester derivatives 11 (4.01g,
50%) as a mixture of diastereoisomers. To a solution
of 11 (4.01g, 15.1mmol) in tetrahydrofuran (8mL)
was added 1M aqueous LiOH (8mL) at 0ꢂC. The reac-
tion mixture was stirred at room temperature 1h, tetra-
hydrofuran was removed in vacuo and the solution was
acidified to pH2 by using 30% aqueous HCl. The solu-
tion was extracted with ethyl acetate (3 · 60mL), the
combined organic phase was dried over sodium sulfate,
and evaporated to dryness. The residue was purified
over silica gel (dichloromethane/acetic acid 49:1) to give
carboxylic acid derivative 12 (2.5g, 65%) as a major iso-
mer. 1H NMR (CDCl3): 7.33 (m, 5H), 5.28 (t,
J = 3.9Hz, 1H), 4.62 (s, 2H), 4.13 (m, 2H), 3.63 (dd,
J = 1, 3.9Hz, 2H), 1.51 (d, J = 5.8Hz, 3H).
The relative energies of northern- and southern-type
conformers for 26a and 35 were calculated and com-
pared to that of the unsubstituted derivative. Nucleoside
conformational preferences were calculated by molecu-
lar mechanics using the MMFFs force field and a dielec-
tric constant of 50. For each nucleoside, 1000
conformers were generated using the JG distance geo-
metry program29 and minimized to low gradient using
BatchMin.30 To estimate the barrier to interconversion
between the northern and southern conformers and
the steepness of the minima, the conformers generated
by distance geometry were also subjected to constrained
minimization, holding one of the ribose ring dihedral
angles at a random value between ꢀ42ꢂ and +42ꢂ by
means
of
a
harmonic
force
constant
of
1000kJmolꢀ1 radꢀ2
.
4.3. Assay for inhibition of HCV replication
Inhibition of HCV replication was evaluated in a subge-
nomic replicon harbored in HB-1 cells as previously
described.17
4.7. (2R,4RS,5R)-4-Acetoxy-2-benzyloxymethyl-5-methyl-
1,3-dioxolane (14)
4.4. Assay for inhibition of HIV spread
To a pre-cooled solution of compound 12 (2.50g,
9.92mmol) and pyridine (1.13g, 14.3mmol) in acetonit-
rile (70mL) was added lead tetraacetate (5.50g,
12.4mmol) portion wise over a period of 10min. The
reaction mixture was stirred at room temperature over-
night. The white precipitate was filtered and the filtrate
was evaporated to dryness. The residue was partitioned
between ethyl acetate (200mL) and saturated aqueous
sodium bicarbonate (150mL). The organic phase was
washed with brine, dried over sodium sulfate, and evap-
orated to dryness. The residue was purified over silica
gel (ethyl acetate/hexane 1:3) to give 14 (1.01g, 38%)
as oil. 1H NMR (CDCl3) (major isomer): 7.33 (m,
5H), 5.93 (d, J = 2.6Hz, 1H), 5.33 (t, J = 4Hz, 1H),
4.61 (s, 2H), 4.18 (m, 1H), 3.62 (d, J = 4.4Hz, 2H),
2.09 (s, 3H), 1.35 (d, J = 6.4Hz, 3H), LRMS-FAB
(M+Li)+ 273.1.
Inhibition of viral spread assays were conducted using
methods previously published.28 Briefly, MT4 human
T-lymphoid cells were infected at a multiplicity of infec-
tion of ꢁ0.01, incubated overnight then washed exten-
sively and plated into 96 well plates. Test compounds
were diluted by twofold serial dilutions and mixed with
the cells. Cultures were incubated an additional 72h and
then assayed for viral production by a commercial HIV
viral core p24 assay kit (Coulter Immunology). End-
point titers were recorded as the compound dilution in
which 95% or greater of the viral antigen production
was inhibited as compared to untreated viral growth
control wells.
4.5. Single-cycle vaccinia infectivity assay
HeLa cells were maintained at 37ꢂC/5% CO2 in phenol
red-free DulbeccoÕs modified EagleÕs Medium media
(InVitrogen) containing 10% heat-inactivated fetal bo-
vine serum (Hyclone) and 1X penicillin–streptomycin
(InVitrogen). For vaccinia infection, cells were seeded
in Costar 3917 plates at 5 · 103 cells/well. After over-
night incubation, the medium was removed and the cells
were infected in the presence or absence of test com-
pounds with 500pfu/well recombinant vaccinia virus
Venv-5.31 This virus expresses b-galactosidase under
the control of the vaccinia 11K promoter.32 After 48h,
b-galactosidase expression was detected using the Gal-
Screen kit (Tropix) according to the manufacturerÕs
instructions.
4.8. (2S,4R,5S)-2-Benzyloxymethyl-5-methyl 1,3-dioxo-
lane-4-carboxylic acid (17) and (2R,4R,5S)-2-benzyloxy-
methyl-5-methyl-1,3-dioxolane-4-carboxylic acid (18)
To a solution of methyl-(4R,5S)-2,2,5-trimethyl-1,3-
dioxolane-4-carboxylate 15 (3.58g, 23.2mmol) and ben-
zyloxyacetaldehyde 9 (4.23g, 23.2mmol) in toluene
(50mL) was added para-toluenesulfonic acid (0.25g,
1.33mmol). The reaction mixture was heated at 80ꢂC
under vacuum for 1h. The reaction mixture was cooled
down to room temperature, diluted with ethyl acetate
(150mL), and washed with saturated sodium bicarbo-
nate (150mL). The organic phase was dried over sodium
sulfate, evaporated to dryness, and purified over silica
gel (ethyl acetate/hexane 1:9) to give the ester derivatives
16 (3.8g, 57%) as a mixture of diastereoisomers.
4.6. (2R,4S,5R)-2-Benzyloxymethyl-5-methyl-1,3-dioxo-
lane-4-carboxylic acid (12)
To a solution of 16 (3.8g, 14.28mmol) in tetrahydrofu-
ran (15mL) was added aqueous LiOH (1M, 15mL)
dropwise at 0ꢂC. The reaction mixture was stirred at
To a solution of (4S,5R)-methyl-2,2,5-trimethyl-1,3-
dioxolane-4-carboxylate 10 (5.25g, 30.1mmol) and ben-

6244
S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
room temperature for 1h. The pH of the solution was
adjusted to 2–3 by addition of 1N aqueous HCl and ex-
tracted with ethyl acetate (3 · 100mL). The combined
organic phase was washed with brine, dried over
Na2SO4, and evaporated to dryness. The residue was
purified over silica gel (dichloromethane–acetic acid
49:1) to give 17 (2.8g, 78%) and 18 (0.45g, 12%). Com-
pound 17: 1H NMR(CDCl3): d 7.33 (m, 5H), 5.97 (br s,
1H), 5.28 (t, J = 3.9Hz, 1H), 4.62 (s, 2H), 4.07–4.21 (m,
2H), 3.64 (dd, J = 0.8, 3.8Hz, 2H), 1.51 (d, J = 5.8Hz,
3H). Compound 18: 1H NMR(CDCl3): d 7.35 (m,
5H), 5.39 (m, 1H), 4.69 (d, J = 6.6Hz, 2H), 4.60 (m,
1H), 4.27 (d, J = 2.8Hz, 1H), 3.75 (m, 2H), 1.39 (d,
J = 6.4Hz, 3H).
poured into a stirred mixture of dichloromethane
(100mL) and saturated aqueous sodium bicarbonate
(100mL), the organic phase was separated, dried over
magnesium sulfate, and evaporated in vacuo. The crude
product was purified on silica gel using hexane/ethyl ace-
tate (1:1 through 1:2) to give the desired diastereoiso-
meric compounds 23a and 24a (0.18g, 30% and 0.12g,
20%, respectively) as colorless oils.
To the individual compounds (0.18g, 0.30mmol and
0.12g, 0.20mmol, respectively) in tetrahydrofuran
(10mL) was added hydrazine hydrate (0.50mL). The
resulting solutions were stirred under reflux for 5h, then
evaporated in vacuo and the crude products purified on
silica gel using dichloromethane/methanol (9:1) as the
eluent. Fractions containing the products were pooled
and evaporated in vacuo to give the desired compounds
(0.113 and 0.080g, respectively) as white powder.
4.9. (2S,4RS,5S)-4-Acetoxy-2-benzyloxymethyl-5-methyl-
1,3-dioxolane (19)
To a solution of 17 (1.76g, 6.98mmol) in acetonitrile
(60mL) were added pyridine (0.88mL) and lead tetraac-
etate (3.87g, 8.73mmol) portion wise over a period of
10min. The reaction mixture was stirred at room tem-
perature overnight. The white precipitate was filtered
and the filtrate was evaporated to dryness. The residue
was partitioned between ethyl acetate (200mL) and sat-
urated aqueous sodium bicarbonate (150mL). The or-
ganic phase was washed with brine, dried over sodium
sulfate, and evaporated to dryness. The residue was
purified over silica gel (ethyl acetate/hexane 1:9) to give
To a solution of the individual compounds (0.113 and
0.08g, respectively) in methanol (4mL) was added palla-
dium hydroxide (10–15mg). The mixtures were stirred
under hydrogen for 24h, filtered through Celite and
evaporated in vacuo. The crude products were purified
on silica gel using dichloromethane/methanol (4:1) as
the eluent. Fractions containing the products were
pooled and evaporated in vacuo to give the desired com-
pounds 25a and 26a (0.044g, 55% and 0.029g, 54%,
respectively) as white powder. Compound 25a: 1H
NMR (200MHz, methanol-d4): 7.89 (s, 1H), 5.73 (d,
J = 5.6Hz, 1H), 5.59 (t, J = 3Hz, 1H), 4.71 (m, 1H),
3.66 (d, J = 3.2Hz, 2H), 1.43 (d, J = 6.2Hz, 3H),
HRMS-FAB (M+H)+ calcd for C10H14N5O4 268.1045,
found 268.1042; 26a: 1H NMR (200MHz, methanol-
d4): d 8.21 (d, 1H), 6.21 (d, 1H), 5.06 (t, 1H), 4.40 (m,
1H), 3.85 (m, 2H), 0.99 (d, 3H), HRMS-FAB (M+H)+
calcd for C10H14N5O4 268.1045, found 268.1044.
1
19 (1.04g, 56%) as oil. H NMR (CDCl3): d 7.34 (m,
5H), 5.93 (d, J = 2.6Hz, 1H), 5.33 (t, J = 4Hz, 1H),
4.61 (s, 2H), 4.18 (m, 1H), 3.62 (d, J = 3.8Hz, 2H),
2.09 (s, 3H), 1.35 (d, J = 6.4Hz, 3H); other isomer:
7.34 (m, 5H), 6.21 (d, J = 3.4Hz, 1H), 5.23 (t,
J = 4.2Hz, 1H), 4.62 (s, 2H), 4.14 (m, 1H), 3.61 (d,
J = 4Hz, 2H), 2.04 (s, 3H), 1.33 (d, J = 6.4Hz, 3H),
LRMS-FAB (M+Li)+ 273.1.
4.10. (2R,4RS,5S)-4-Acetoxy-2-benzyloxymethyl-5-meth-
yl-1,3-dioxolane (20)
4.12. (2R,4S,5R)-2,6-Diamino-9-[2-hydroxymethyl-5-
methyl-1,3-dioxolan-4-yl]purine (25b) and (2R,4R,5R)-
2,6-diamino-9-[2-hydroxymethyl-5-methyl-1,3-dioxolan-
4-yl]purine (26b)
Compound 20 was obtained 55% yield from 18 follow-
ing the procedure described for 19. H NMR (CDCl3):
1
d 7.33 (m, 5H), 5.99 (s, 1H), 5.47 (m, 1H), 4.61 (m,
2H), 4.38 (m, 1H), 3.58 (d, J = 4.6Hz, 2H), 2.02
(s, 3H), 1.28 (d, J = 6.6Hz, 3H); minor isomer: 7.33
(m, 5H), 6.31 (d, J = 3.6Hz, 1H), 5.47 (m, 1H), 4.61
(m, 2H), 4.38 (m, 1H), 3.53 (d, J = 3.8Hz, 2H), 2.11
(s, 3H), 1.28 (d, J = 6.6Hz, 3H), LCMS (M+H)+ 267.2.
To
a
pre-cooled (0ꢂC) solution of 14 (0.266g,
1.00mmol) in dichloromethane (5mL) was added
bromotrimethylsilane (0.46g, 3.00mmol). The resulting
solution was stirred at 0ꢂC for 15min, then at room
temperature for 15min. The solution was evaporated
in vacuo and co-evaporated twice from acetonitrile.
The residue was taken up in acetonitrile (5mL) and
added to a vigorously stirred mixture of 2-amino-6-chloro-
purine 22, powdered KOH, and tris-[2-(2-methoxyeth-
oxy)ethyl]amine in acetonitrile (5mL). The mixture was
stirred for 20min at room temperature and poured into
a stirred mixture of diethyl ether (100mL) and saturated
aqueous bicarbonate (100mL). The organic phase was
separated, washed once with water (100mL), dried over
magnesium sulfate, and evaporated under vacuo. The
residue was purified on silica gel using ethyl acetate/hex-
ane (1:1) as the eluent. Fractions containing the product
were pooled and evaporated under vacuo to give the de-
sired compound (0.065g) as a inseparable mixture of
diastereoisomers 23b and 24b (0.065g, 17.3%).
4.11. (2R,4S,5R)-9-[2-Hydroxymethyl-5-methyl-1,3-di-
oxolan-4-yl]guanine (25a) and (2R,4R,5R)-9-[2-hydroxy-
methyl-5-methyl-1,3-dioxolan-4-yl]guanine (26a)
To diphenylcarbamic acid 2-acetylamino-9H-purin-6-yl
ester 21 (0.266g, 1.00mmol) in dichloroethane (8mL)
was
added
bis-(trimethylsilyl)acetamide
(0.81g,
4.00mmol) at rt. The mixture was stirred at room tem-
perature for 2h, then the acetate derivative 14 (0.27g,
1.00mmol) in dichloroethane (3mL), and then dropwise
trimethylsilyl triflate (0.23g, 1.05mmol), was added. The
resulting mixture was stirred at rt for 30min and then
heated to reflux overnight. The reaction mixture was

S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
6245
A solution of 23b and 24b (0.065g, 0.17mmol) in meth-
anolic ammonia (4mL) was heated in a closed container
overnight at 55ꢂC. The mixture was cooled and evapo-
rated in vacuo. The residue was purified on silica gel
using methanol/dichloromethane (1:9) as the eluent.
Fractions containing the product were pooled and evap-
orate in vacuo to give the desired 2,6-diamino intermedi-
ate (0.037g) as a mixture of diastereoisomers. To this
compound in methanol (12mL) was added ammonium
formate (0.04g) and Pd/C (10%) (0.040g). The resulting
mixture was heated under reflux for 2h. The mixture
was allowed to come to room temperature, filtered
through Celite and evaporated under vacuo. The residue
was purified on silica gel using methanol/dichlorometh-
ane (1:4) as the eluent. Fractions containing the product
were pooled and evaporated in vacuo to give the desired
compound (0.017g, 34% in two steps) as a mixture of
diastereoisomers 25b and 26b. A fraction of the diastereo-
isomeric mixtures (0.013g) were separated by HPLC
[column: ChiralPak AD (4.6mm · 250mm · 10l); flow:
1mL/min; eluent: 100% EtOH; detection 250nm] to give
Following the same procedure as described for 30a,
the cis derivative 29a (0.028g) was converted to 31a
(0.008g, 39% in two steps). Compound 30a: 1H
NMR(200MHz, methanol-d4): d 7.68 (s, 1H), 5.70 (d,
J = 5.6Hz, 1H), 5.55 (t, J = 3.2Hz, 1H), 4.68 (m, 1H),
3.62 (d, J = 3.4Hz, 2H), 1.39 (d, J = 6.2Hz, 3H),
HRMS-FAB (M+H)+ calcd for C10H14N5O4 268.1045,
1
found 268.1047; 31a: H NMR (200MHz, d4-MeOH):
d 8.21 (s, 1H), 6.21 (d, J = 4.8Hz, 1H), 5.06 (t,
J = 2.2Hz, 1H), 4.40 (m, 1H), 3.85 (m, 2H), 1.00 (d,
J = 6.2Hz, 3H), HRMS-FAB (M+H)+ calcd for
C10H14N5O4 268.1045, found 268.1049.
4.14. (2S,4R,5S)-6-Amino-9-[2-hydroxymethyl-5-methyl-
1,3-dioxolan-4-yl]purine (30b) and (2S,4S,5S)-6-amino-9-
[2-hydroxymethyl-5-methyl-1,3-dioxolan-4-yl]purine (31b)
To a solution of 19 (0.21g, 0.79mmol) in dichlorometh-
ane (4mL) was added bromotrimethylsilane (0.21mL,
1.6mmol) at 0ꢂC. The reaction mixture was stirred at
0ꢂC for 30min and at room temperature for 45min. Sol-
vent was evaporated, co-evaporated with acetonitrile.
The residue was dissolved in acetonitrile and added to
a vigorously stirred solution of 6-chloropurine 27
(0.125g, 0.8mmol), KOH (0.13g), and tris-[2-(2-meth-
oxyethoxy)ethyl]amine (0.05mL) in acetonitrile (4mL).
The reaction mixture was stirred at room temperature
for 45min. The solution was taken in ethyl acetate
(100mL) and washed with water (100mL) and brine
(100mL). The organic phase was dried over sodium
sulfate, evaporated to dryness and purified over silica
gel to give an inseparable mixture 28b and 29b.
(0.086g, 30%).
1
25b (0.0045g) and 26b (0.0018g). Compound 25b: H
NMR (200MHz, DMSO-d6): d 7.89 (s, 1H), 6.69 (s,
2H), 5.79 (s, 2H), 5.61 (d, J = 5.8Hz, 1H), 5.41 (t,
J = 3.6Hz, 1H), 4.95 (br s, 1H), 4.71 (m, 1H), 3.43 (m,
2H), 1.25 (d, J = 6.2Hz, 3H), HRMS-FAB (M+H)+
calcd for C10H15N6O3 267.1205, found 267.1211; 26b:
1H NMR (200MHz, d6-DMSO): d 7.92 (s, 1H), 6.68
(s, 2H), 6.05 (d, J = 4.8Hz, 1H), 5.76 (s, 2H), 5.27 (br
s, 1H), 4.94 (m, 1H), 4.33 (m, 1H), 3.66 (m, 2H), 0.81
(d, J = 6.2Hz, 3H), HRMS-FAB (M+H)+ calcd for
C10H15N6O3 267.1205, found 267.1209.
4.13. (2S,4R,5S)-9-[12-Hydroxymethyl-5-methyl-1,3-di-
oxolan-4-yl]guanine (30a) and (2S,4S,5S)-9-[2-hydroxy-
methyl-5-methyl-1,3-dioxolan-4-yl]guanine (31a)
A solution of a mixture of 28b and 29b (0.075g,
0.21mmol) in dioxane (3mL) and liquid ammonia
(8mL) was heated at 80ꢂC in a steel bomb overnight.
The reaction mixture was cooled down, evaporated to
dryness, and purified over silica gel to give a diastereo-
isomeric mixture of adenine derivatives, which was dis-
solved in ethanol (10mL) and cyclohexene (5mL). To
this solution was added palladium hydroxide on char-
coal (20% Pd, 0.05g) and the suspension was under re-
flux for 5h. The reaction mixture was filtered, the
filtrate was evaporated, and the residue was purified
over silica gel (dichloromethane/methanol 47:3) to give
inseparable mixture of 30b and 31b (0.036g, 69% in
two steps). A fraction of the diastereoisomeric mixtures
(0.019g) were separated by HPLC [column: Chiralcel OJ
(2cm · 25cm · 10l); flow: 1mL/min; eluent: 20%
EtOH/hexane; detection 260nm] to give 30b (0.0055g)
A suspension of 21 (0.24g, 0.61mmol) and bis-(trimeth-
ylsilyl)acetamide (0.6mL, 2.44mmol) in 1,2-dichloro-
ethane (8mL) was stirred at room temperature for
1.5h. To this clean solution was added 19 (0.11g,
0.41mmol) in 1,2-dichloroethane (5mL) followed by tri-
methylsilyl triflate (0.086mL, 0.47mmol). The reaction
mixture was heated under reflux for 4h. The solution
was poured into saturated aqueous sodium bicarbonate
and extracted with dichloromethane (2 · 50mL). The
combined organic phase was dried over sodium sulfate,
evaporated, and the residue was purified over silica gel
(ethyl acetate/hexane 1:1) to give 28a (0.031g, 13%)
and 29a (0.03g, 12.5%).
To a solution of 28a (0.028g, 0.077mmol) in tetra-
hydrofuran (4mL) was added hydrazine hydrate and
the solution was refluxed for 3h. The solvent was evap-
orated under reduced pressure and the residue was puri-
fied over silica gel to give the guanine derivative. This
was dissolved in ethanol (5mL) and cyclohexene
(2.5mL) and to this solution was added palladium
hydroxide on charcoal (20% Pd, 0.025g). The suspen-
sion was heated under reflux for 5h, cooled down and
filtered. The filtrate was evaporated to dryness and the
residue was purified over silica gel (dichloromethane/
methanol 9:1) to give 30a (0.006g, 29% in two steps).
1
and 31b (0.011g) as white powder. Compound 30b: H
NMR (200MHz, methanol-d4): d 8.31 (s, 1H), 8.20 (s,
1H), 5.91 (d, J = 5.4Hz, 1H), 5.62 (t, J = 3.2Hz, 1H),
4.81 (m, 1H), 3.68 (d, J = 3.2Hz, 2H), 1.45 (d,
J = 6.4Hz, 3H), HRMS-FAB (M+H)+ calcd for
1
C10H14N5O3 252.1096, found 252.1092; 31b: H NMR
(200MHz, methanol-d4): d 8.67 (s, 1H), 8.19 (s, 1H),
6.41 (d, J = 4.8Hz, 1H), 5.12 (t, J = 2Hz, 1H), 4.47
(m, 1H), 3.91 (m, 2H), 0.95 (d, J = 6.4Hz, 3H),
HRMS-FAB (M+H)+ calcd for C10H14N5O3 252.1096,
found 252.1089.

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S. Bera et al. / Bioorg. Med. Chem. 12 (2004) 6237–6247
8. Jeong, L. S.; Schinazi, R. F.; Beach, J. W.; Kim, H. O.;
Shanmuganathan, K.; Nampalli, S.; Chun, M. W.; Chung,
W. K.; Choi, B. G.; Chu, C. K. J. Med. Chem. 1993, 36,
2627.
4.15. (2R,4S,5S)-9-[2-Hydroxymethyl-5-methyl-1,3-di-
oxolan-4-yl]guanine (34) and (2R,4R,5S)-9-[2-hydroxy-
methyl-5-methyl-1,3-dioxolan-4-yl]guanine (35)
9. Grove, K. L.; Guo, X.; Liu, S. H.; Gao, Z.; Chu, C. K.;
Cheng, Y. C. Cancer Res. 1995, 55, 3008.
10. Kim, H. O.; Schinazi, R. F.; Satyanarayana, N.; Shanm-
uganathan, K.; Cannon, D. L.; Alves, A. J.; Jeong, L. S.;
Beach, J. W.; Chang, C. N.; Chu, C. K. J. Med. Chem.
1993, 36, 30.
11. Furman, P. A.; Jeffrey, J.; Kiefer, L. L.; Feng, J. Y.;
Anderson, K. S.; Borroto-Esoda, K.; Hill, E.; Copeland,
W. C.; Chu, C. K.; Sommadossi, J.-S.; Liberman, I.;
Scinazi, R. F.; Painter, G. R. Antimicrob. Agents Chem-
other. 2001, 45, 158.
12. Chin, R.; Shaw, T.; Torresi, J.; Sozzi, V.; Trautwein, C.;
Block, T.; Manns, M.; Isom, H.; Furman, P.; Locarnini, S.
Antimicrob. Agents Chemother. 2001, 45, 2495.
13. Jeong, L. S.; Schinazi, R. F.; Beach, J. W.; Kim, H. O.;
Nampalli, S.; Shanmuganathan, K.; Alves, A. J.; McMil-
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181.
A suspension of 21 (0.485g, 1.25mmol) and BSA
(0.7mL, 2.84mmol) in 1,2-dichloroethane (8mL) was
stirred at room temperature for 1.5h. To this clean solu-
tion was added 20 (0.19g, 0.71mmol) in 1,2-dichloro-
ethane (5mL) followed by trimethylsilyl triflate
(0.15mL, 0.81mmol). The reaction mixture was heated
under reflux for 4h. The solution was poured into satu-
rated aqueous sodium bicarbonate and extracted with
dichloromethane (2 · 80mL). The combined organic
phase was dried over Na2SO4, evaporated and the resi-
due was purified over silica gel (ethyl acetate/hexane:
1/1) to give inseparable mixture (4:3) of a and b nucleo-
side derivatives 32 and 33 (0.08g, 19%). The mixture of
32 and 33 (0.07g, 0.118mmol) in tetrahydrofuran (8mL)
was added hydrazine hydrate (0.4mL) and the reaction
mixture was heated under reflux for 3h. The solution
was evaporated to dryness and the residue was purified
over silica gel (dichloromethane/methanol 93:7) to give
a mixture of nucleoside derivatives, which was dissolved
in ethanol (8mL) and cyclohexene (4mL). To this solu-
tion was added palladium hydroxide on charcoal (20%
Pd, 0.03g) and the suspension was refluxed for 40h.
The reaction mixture was cooled down to room temper-
ature, filtered, and the filtrate was evaporated to dry-
ness. The residue was purified over silica gel
(dichloromethane/methanol 9:1) to give 34 (0.0047g,
15% in two steps) and 35 (0.0065, 21% in two steps).
Compound 34: 1H NMR (200MHz, methanol-d4): d
7.94 (s, 1H), 5.81 (d, J = 2.8Hz, 1H), 5.24 (t,
J = 2.8Hz, 1H), 4.61 (m, 1H), 3.63 (d, J = 2.8Hz, 2H),
1.31 (d, J = 6.6Hz, 3H), HRMS-FAB (M+H)+ calcd
for C10H14N5O4 268.1045, found 268.1049; 35: 1H
NMR (200MHz, methanol-d4): d 7.62 (s, 1H), 6.27 (d,
J = 4.4Hz, 1H), 5.64 (t, J = 3.6Hz, 1H), 4.63 (m, 1H),
3.66 (d, J = 3.8Hz, 2H), 1.02 (d, J = 6.4Hz, 3H),
HRMS-FAB (M+H)+ calcd for C10H14N5O4 268.1045,
found 268.1042.
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