6-Azapyrimidine-2′-deoxy-4′-thionucleosides: Antiviral agents against TK+ and TK- HSV and VZV strains

Article abstract of DOI:10.1021/jm049806q

The synthesis of a series of novel 1-(2-deoxy-4-thio-β-D-erythro- pentofuranosyl)-(6-azapyrimidine) nucleosides is described. X-ray crystallographic data of the thymidine derivative allowed conformational analysis, which indicated a twist (³T

Full text of DOI:10.1021/jm049806q

5482
J . Med. Chem. 2004, 47, 5482-5491
6-Aza p yr im id in e-2′-d eoxy-4′-th ion u cleosid es: An tivir a l Agen ts a ga in st TK+ a n d
TK- HSV a n d VZV Str a in s
Hannah L. Maslen,† David Hughes,‡ Mike Hursthouse,‡ Erik De Clercq,§ J an Balzarini,§ and Claire Simons*,†
Medicinal Chemistry Division, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue,
Cardiff CF10 3XF, UK; EPSRC X-ray Crystallography Service, Department of Chemistry, University of Southampton,
Highfield, Southampton SO17 1BJ , UK; and Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received March 8, 2004
The synthesis of a series of novel 1-(2-deoxy-4-thio-â-D-erythro-pentofuranosyl)-(6-azapyrimidine)
nucleosides is described. X-ray crystallographic data of the thymidine derivative allowed
conformational analysis, which indicated a twist (3T2) sugar conformation. Hydrogen-bonded
assemblies for the crystal structure were determined using PLATON software to allow further
interpretation of the crystal packing and base interactions. The 6-azapyrimidine nucleosides
described were evaluated against a range of viral strains. The thymidine analogue showed
pronounced activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella-
zoster virus (VZV), and vaccinia virus. This compound lost only 5- to 10-fold of its antiviral
activity against thymidine kinase (TK)-deficient HSV-1 and VZV strains. These observations
suggest that the compounds may not entirely depend on viral TK-catalyzed phosphorylation
for antiviral activity and/or use an alternative metabolic activation pathway, and/or display a
unique mechanism of antiviral action by the unmetabolized nucleoside analogue.
In tr od u ction
Both 5- and 6-azapyrimidine nucleosides are of inter-
est owing to their biological properties (Figure 1).1
5-Azacytidine 1 is active against T-4 lymphomas and
L1210 leukaemia. As a result, 5-azacytidine and its 2′-
deoxy derivative 2 are used clinically in the treatment
of acute leukaemia.2,3 5,6-Dihydro-5-azathymidine 3, a
naturally occurring nucleoside antibiotic isolated from
Streptomyces platensis var. clarensis, exhibits in vitro
activity against DNA viruses.4 In the 6-azapyrimidine
nucleoside series, 6-azauridine monophosphate 4 is of
most interest, owing to its antiviral activity which
results from inhibition of orotidine 5′-monophosphate
decarboxylase, an enzyme involved in the de novo
pyrimidine mononucleotide biosynthesis.5
F igu r e 1. Biologically active azapyrimidines.
As with all conventional glycosidic linkages, these
azapyrimidine nucleosides are susceptible to cleavage
by nucleoside phosphorylases. The toxicity associated
with these azapyrimidine nucleosides may well result
from release of the 5- or 6-azapyrimidine base, which
are known to display narcotic activity and cause central
nervous system disturbances in man.6 This toxicity
detracts from the potential of the azapyrimidine nucleo-
sides; however, the demonstrated resistance of 4′-
thionucleosides toward phosphorolytic cleavage7 may
provide a less toxic derivative with clinical potential.
F igu r e 2. Structures of 6-azapyrimidine 2′-deoxy-4′-thio-
nucleosides 5â-7â, 8, and 9.
the HSV-1 thymidine kinase. 5-Substituted pyrimidine
nucleosides often display potent anti-herpes virus activ-
ity;9,10 therefore, it was of interest to prepare 1-(2-deoxy-
4-thio-â-D-erythro-pentofuranosyl)-(5-substituted-6-aza-
pyrimidine) nucleosides (5â-7â, 8, and 9) for biological
evaluation (Figure 2).
1-(2-Deoxy-4-thio-â-D-erythro-pentofuranosyl)-(6-aza-
uracil), which has previously been described,8 was
devoid of antiviral activity. The lack of anti-herpes
activity was proposed to be owing to poor affinity for
Resu lts
Ch em istr y. The 5-methyl-6-azapyrimidine 10 was
commercially available; the 5-ethyl- and 5-propyl-6-
azapyrimidines, 16 and 17 respectively, were prepared
via the semicarbazones, 14 and 15, by the reaction of
semicarbazide hydrochloride 11 with the corresponding
2-keto-carboxylic acids 12 and 13, according to a litera-
ture procedure (Scheme 1).6 A 25 °C discrepancy be-
* To whom correspondence should be addressed. Tel: +44 (0)2920
876307. Fax: +44 (0)2920 874149. E-mail: SimonsC@Cardiff.ac.uk.
† Cardiff University.
‡ University of Southampton.
§ Katholieke Universiteit Leuven.
10.1021/jm049806q CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/29/2004

6-Azapyrimidine-2′-deoxy-4′-thionucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5483
Sch em e 1a
a
Conditions: (a) R ) Et, H2O, 1 h; R ) Pr, H2O, EtOH, 100 °C, 1 h then 4 °C, 12 h. (b) i. NaOEt, EtOH, ethylene glycol, 100 °C, 48-72
h; ii. aqueous HCl (pH 2), 4 °C, 12 h.
Sch em e 2a
a
Conditions: (a) N,O-Bis(trimethylsilyl)acetamide, CH3CN, 1 h. (b) ICl, CH3CN, 4 Å molecular sieves, 24 h. (c) NIS, CH3CN, 4 Å
molecular sieves, 24 h. (d) BCl3, CH2Cl2, -80 °C, 4 h.
Ta ble 1. R:â Ratios from Sugiyama Coupling
tween the literature mp (175-176 °C) and the mp of
compound
R:â ratio
yield, %
the prepared compound 15 (200-202 °C) was noted;
however, 1H and 13C NMR confirmed the purity and
structure of the prepared compound. 13C NMR displayed
characteristic signals for the carboxylic acid, δ ∼ 165,
amide, δ ∼ 157, and NdC, δ ∼ 140. Cyclization of the
semicarbazones 14 and 15 using sodium ethoxide gave
the required 5-alkyl-6-azapyrimidines 16 and 17 in low
yields and, in the case of 17, the reaction failed to reach
completion even after 72 h reaction time with a 1:5
mixture of 13:17 obtained (Scheme 1).
19, R ) Me
20, R ) Et
21, R ) nPr
1:1
1:1
1:1
78
70
76
In an attempt to improve the anomeric ratio of the
coupled nucleosides, the glycal methodology was ap-
plied.13 This involved initial reaction of the thiosugar
18 with iodine monochloride in the presence of 2,6-di-
tert-butyl-4-methylpyridine at 0 °C for 5 h to form the
glycal 22 (Scheme 3). Introduction of the silyl protected
azapyrimidine bases and an additional quantity of
iodine monochloride resulted in formation of the 2-iodo-
nucleosides 23-25 in reasonable yields (Scheme 3). As
can be seen from Table 2, improved R:â ratios were
obtained; however, this promising ratio was negated by
the unreliability of the radical deiodination step which,
in our hands, resulted in complex mixtures. Owing to
the unsatisfactory deiodination reaction, the Sugiyama
method (Scheme 2) would appear to provide the opti-
mum coupling procedure for these nucleosides.
The benzyl-protected 2′-deoxy-4′-thionucleosides 19-
21 were prepared by the reaction of the thiosugar 1811
with the bis-silylated 6-azapyrimidine bases, prepared
by the reaction of 10, 16, and 17 with N,O-bis(trimethyl-
silyl)acetamide in anhydrous acetonitrile for 1 h, in the
presence of either iodine monochloride or N-iodosuccin-
imide (NIS) (Scheme 2). The use of iodine reagents as
coupling catalysts was based on the procedure described
by Kim and Misco12 for the preparation of 4′-thionucleo-
sides (Sugiyama coupling method), which results in
approximately 1:1 ratios of the R- and â-anomers in
reasonable yields (Table 1). Debenzylation of 19-21
with boron trichloride allowed the separation of the
anomers (Scheme 2).
Further modification of the base moiety involved
preparation of the C-4 thiocarbonyl derivative 8, which
was subsequently converted to the 6-aza-5-methyl-

5484 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Maslen et al.
Sch em e 3a
a
Conditions: (a) ICl, CH2Cl2, 2,6-di-tert-butyl-4-methylpyridine, 0 °C, 5 h. (b) i. (6-azapyrimidine)TMS2, CH2Cl2; ii. ICl, CH2Cl2, 16 h.
(c) azobiscyclohexane carbonitrile, Bu3SnH, toluene, 45-60 °C.
Ta ble 2. R:â Ratios from Glycal Coupling
Ta ble 3. 1H NMR Data for Compounds 5-9
compound
R:â ratio
yield, %
compound
H-1′
J 1′,2′
H-2′ endo H-2′ exo
∆
2′endo,2′exo
23, R ) Me
24, R ) Et
25, R ) nPr
1:4
1:3
1:3
70
67
67
5Ra
5âa
6Rb
6âb
7Ra
7âa
8a
6.34, dd 5.9, 8.1 2.65, m
6.37, dd 5.1, 7.3 2.70, m
2.51, m
2.35, m
0.14
0.35
-
0.33
0.12
0.28
0.32
0.35
6.13, t
7.7
2.49, mc
6.19, ψt 5.5, 6.8 2.50, mc
6.13, dd 5.9, 8.2 2.73, mc
6.30, dd 4.7, 7.3 2.57, mc
6.29, dd 5.0, 7.3 2.69, m
6.21, ψt 6.0, 6.7 2.46, m
2.17, m
2.61, m
2.29, m
2.37, m
2.11, m
cytosine nucleoside 9 using conventional methodology
(Scheme 4). Preparation of 8 required initial acetyl
protection of 1-(2-deoxy-4-thio-â-D-erythro-pentofurano-
syl)-(6-azathymine) 5â. The acetylated nucleoside 26
was then treated with Lawesson’s reagent14 to give 27.
Reaction of 27 with methanolic ammonia at 0 °C and
then increasing to room temperature gave the de-
acetylated C-4 thiocarbonyl nucleoside 8. Reaction of 27
with methanolic ammonia at 30 °C in a sealed vessel
for 72 h gave the deacetylated C-4 amino nucleoside 9
(Scheme 4).
9b
CD3OD. DMSO-d6. c Overlapping with CH2 from Et/Pr sig-
a
b
nal.
between the anomers from the chemical shift of H-1′
with H-1′â occurring further downfield than H-1′R. The
H-2′ signal splitting patterns were very distinctive with
the two multiplets for H-2′R (endo and exo) separated
by approximately 0.1 ppm, whereas the two multiplets
for H-2′â were separated by approximately 0.3 ppm.
This characteristic H-2′ splitting pattern has previously
been observed for 2′-deoxy-4′-thio-nucleosides.8,15
Confirmation of anomeric configuration was obtained
by H NMR (Table 3) and, in the case of 5, by X-ray
crystallography (Figure 2). It was possible to distinguish
1
Sch em e 4a
a
Conditions: (a) Ac2O, pyridine, 16 h. (b) Lawesson’s reagent, toluene, reflux, 3 h. (c) NH3/MeOH, 0 °C, 4 h then room temperature,
4 h. (d) NH3/MeOH, 30 °C, sealed vessel, 72 h.

6-Azapyrimidine-2′-deoxy-4′-thionucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5485
cytomegalovirus (strains AD169 and Davis) and vari-
cella-zoster virus (strains YS and OKA) as well as the
thymidine kinase (TK) deficient 07/1 and YS/R VZV
strains (Table 5). Compound 5â displayed activity
comparable with acyclovir (ACV) (EC50 ∼ 1 µM); how-
ever, it was considerably less active than brivudin ((E)-
5-(2-bromovinyl)-2′-deoxyuridine, BVDU) (EC50: 0.003
µM). However, importantly 5â showed a reasonably good
retention of antiviral activity in VZV/TK- strains,
whereas BVDU lost activity against TK- VZV strains
by at least 4 orders of magnitude. It should also be
mentioned that 5â was inhibitory to both HSV-1 and
HSV-2, whereas BVDU selectively inhibited HSV-1, but
not HSV-2.
F igu r e 3. ORTEP diagram of 5â.
Compound 5â did not inhibit CMV replication in cell
culture at 200 µM. The other compounds, 6â, 7â, 8, 9,
and 27, were also devoid of anti-VZV/CMV activity at
40 µM.
Compound 5â was also found to display pronounced
activity against HSV-1 (strain KOS) and HSV-2 (strain
G), with comparable EC50 values (1.3-3.2 µg/mL) (Table
6). However, the antiviral potential of these compounds
were inferior to those of acyclovir and BVDU. Interest-
ingly, 5â only lost its anti-HSV-1 potential by 5- to 10-
fold against a TK-deficient HSV-1 strain. An interesting
antiviral activity was also noted for 5â against vaccinia
virus (EC50: 2.2 µg/mL).
The compounds were also evaluated against a broad
range of other viruses, including reovirus-1, Coxsackie
virus B4, Sindbis virus, parainfluenza-3 virus, Punta
Toro virus, vesicular stomatitis virus, and respiratory
syncytial virus, but were found to be inactive at subtoxic
concentrations.
Ta ble 4. Selected Geometrical Parameters for Compound 5â
Compared with 2′-Deoxy-4′-thiothymidine
5â
4′S-T (II)17
Bond Lengths (Å)
1.516 (2)
C1′-C2′
C2′-C3′
C3′-C4′
C1′-S4′
C4′-S4′
C1′-N1
1.525 (4)
1.508 (4)
1.545 (4)
1.839 (3)
1.831 (3)
1.480 (4)
1.523 (2)
1.527 (2)
1.838 (15)
1.825 (16)
1.469 (19)
Bond Angles (deg)
108.41 (13)
107.63 (13)
106.12 (11)
94.87 (7)
C1′C2′C3′
C2′C3′C4′
C3′C4′S4′
C4′S4′C1′
S4′C1′C2′
S4′C1′N1
106.2 (3)
106.6 (2)
105.4 (2)
94.0 (1)
105.9 (2)
113.5 (2)
106.24 (11)
111.00 (10)
Torsion Angles (deg)
10.37 (11)
C4′S4′C1′C2′ (υ0)
S4′C1′C2′C3′ (υ1)
C1′C2′C3′C4′ (υ2)
C2′C3′C4′S4′ (υ3)
C3′C4′S4′C1′ (υ4)
O5′C5′C4′C3′ (γ)
C5′C4′C3′O3′ (δ)
S4′C1′N1C2 (ø)
S4′C1′N1N6
-14.5 (2)
39.0 (3)
-50.4 (3)
37.9 (3)
-13.0 (3)
179.0 (2)
-
-32.52 (14)
44.58 (16)
-35.03 (15)
14.11 (12)
65.34 (17)
78.39 (15)
-76.04 (15)
89.64 (14)
Discu ssion
Compound 5â displayed a C3′-endo/2′-exo twist (North
3
-144.2 (3)
-
type, T2) sugar conformation, which has been shown
to be a favorable conformation with respect to biological
activity from studies employing the rigid methano-
carbocyclic nucleosides.19 As noted in Table 4, both 5â
and 4′S-T adopt the anti conformation. However, analy-
sis of the torsion angle data for the ø(S-C1′-N1-C2)
angle reveals a value of -76.0° for 5â and -144.2 for
4′S-T. To investigate the origins of this conformational
difference the Hydrogen-bonded assemblies for both
structures were determined using PLATON software.20
Inspection of Figure 4 and correlation with the
determined hydrogen bonded assemblies allows us to
make the following statements:
• Both structures contain the common medium
strength intermolecular interaction, H3‚‚‚O3′.
• Both O5′ and O4 interact with hydroxyl (OH)
hydrogens in both structures, but with different ones,
to form medium strength intermolecular interactions.
• Both structures contain further weak intermolecular
interactions of equal strength, H7C‚‚‚O2 and H1′‚‚‚O4
in 5â, H2′B‚‚‚O2 and H4′‚‚‚O4 in 4′ S-T.
• Both structures contain the common intramolecular
H1′‚‚‚O2 interaction. Again, this bond is roughly of equal
strength.
• The presence of the hydrogen-bond acceptor N6 in
5â is contrasted to the presence of a C-H6 hydrogen-
bond donor in 4′ S-T.
Pseudorotational Parameters (deg)
phase angle (P)
2.608
177.7
47.9
degree of pucker (υm)
sugar conformation
44.626
3T2
2E
glycosidic conformation
C4′-C5′ conformation
anti
anti
trans (ap)
gauche g+ (+sc)
Con for m a tion a l An a lysis. X-ray crystallography
provided absolute confirmation of the anomeric config-
uration of the 6-aza-thymidine nucleosides 5â (Figure
3).
Selected geometrical parameters16 for 5â are de-
scribed in Table 4, and compared with 2′-deoxy-4′-
thiothymidine (4′S-T),17 a known substrate for viral
thymidine kinase which displays activity against herpes
simplex virus (HSV).18
The X-ray crystallographic data indicated an anti
conformation (torsional angle ø ) -76.04°). The major
differences between 5â and 4′S-T were the conformation
about the C4′-C5′ bond, with a gauche (g+ or +sc)
orientation for 5â compared with trans (or ap) orienta-
tion for 4′S-T, and the ring puckering with 5â displaying
a North-type (3T2) and 4′S-T a South-type (2E) confor-
mation.
An tivir a l Activity. The antiviral effects of com-
pounds 5â, 6â, 7, 8, 9, and 27 were evaluated against

5486 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Maslen et al.
Ta ble 5. Activity of Compounds against Varicella-zoster Virus (VZV) and Cytomegalovirus (CMV) in Human Embryonic Lung (HEL)
Cell Cultures
a
antiviral activity EC50 (µM)
TK+ VZV
TK- VZV
CMV
AD169 strain
compound
YS strain
4.1
>40
>40
>40
>40
>40
OKA strain
07/1 strain
YS/R strain
Davis strain
MCCb µM
(CC50)c µM
5â
2.5
>40
>40
>40
>40
>40
0.9
14
>40
>40
>40
>40
>40
24
g5d
>40
>40
>40
>40
>40
27
>200
>40
>200
>40
>40
>40
>40
>40
>400
>400
1.5
>200
>40
>40
>40
>40
>40
-
166
>40
>40
>40
>40
>40
-
6â
7â
>40
8
9
>40
>40
27
>40
acyclovir
brivudin
ganciclovir
1.3
0.003
-
>200
>150
0.003
-
29
-
56
-
-
>150
-
>150
0.5
a
b
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque forming units (PFU). Minimum
cytotoxic concentration that causes microscopically detectable alteration of cell morphology. c Cytotoxic concentration required to reduce
d
cell growth by 50%. No complete inhibition at higher drug concentrations.
Ta ble 6. Activity of Compounds against Herpes Simplex Virus and Vaccinia Virus in E6SM Cell Cultures
antiviral activity EC50 (µg/mL)a
compound
HSV-1 (KOS)
HSV-2 (G)
HSV-1 TK- (KOS ACVr)
Vaccinia virus
MCCb (µg/mL)
5â
1.3
120
>200
120
24
>8
0.015
0.076
0.004
3.2
>200
>200
120
24
>8
80
18
>200
120
120
120
>8
2.2
>200
>200
120
24
>8
0.384
>400
>100
>200
>200
>200
>200
>200
g 40
>400
>400
>100
6â
7â
8
9
27
brivudin
acyclovir
ganciclovir
16
0.076
0.003
48
2.4
a
b
Effective concentration required to reduce virus plaque formation by 50%. Virus input was 20 plaque forming units (PFU). Minimum
cytotoxic concentration that caused a microscopically detectable alteration of cell morphology.
F igu r e 4. Both analogues contain the intramolecular bond, C1′-H1′‚‚‚O2. However, the “scorpion-like” conformation of 5â may
be attributed to the intramolecular C3′-H3′‚‚‚N6 bond.
In summary, therefore, it is proposed that the forces
acting upon 5â and 4′ S-T appear to be equal in terms
of medium strength and weak intermolecular consid-
erations. These structures even have one intramolecular
interaction in common. The difference, however, lies in
the presence of an intramolecular C-H3′‚‚‚N6 bond in
5â that is absent from 4′ S-T. It is this bond, therefore,
that is thought to be responsible for the “scorpion-like”
conformation noted in 5â and shown in Figure 4.
preferences in nucleotide bases. In this case it was
suggested that specific C-H‚‚‚O interactions between
the purine C8-H or pyrimidine C6-H and the oxygen O5′
are responsible for the anti conformation of these bases.
Further evidence for the structural significance of this
bond lies in the presence of the gauche conformation in
the nucleoside ring of 5â. Such conformational prefer-
ences are thought to be responsible for the packing
differences found between 5â and 4′ S-T.
This situation is analogous to that found in nucleo-
tides by Rubin et al.21 to explain the conformational
Biologically 5â and 4′ S-T both display antiviral
activity vs herpes simplex virus; however, 5â has a

6-Azapyrimidine-2′-deoxy-4′-thionucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5487
mmol) in small portions. The suspension was heated to 100
°C, and ethanol (40 mL) was added in order to form a solution.
The reaction was cooled to room temperature and then left in
a refrigerator (4 °C) for 12 h. The resulting solid was filtered,
washed with cold water, and dried under vacuum at 70 °C for
12 h to give 11.42 g (91%) of 15 as a white crystalline solid:
mp: 200-202 °C (lit. mp: 175-176 °C); 1H NMR (DMSO-d6):
δ 11.85 (s, 1, OH), 10.17 (s, 1, NH), 7.38 (bs, 1, NH2), 6.68 (bs,
1, NH2), 2.50 (t, 2, J ) 7.6 Hz, CH2), 1.35 (m, 2, J ) 7.6 Hz, J
) 7.3 Hz, CH2), 0.87 (t, 3, J ) 7.3 Hz, CH3). 13C NMR (DMSO-
d6): δ 165.10 (CdO, acid), 156.76 (CdO, amide), 139.59 (Cq),
26.32 (CH2), 19.09 (CH2), 13.98 (CH3).
5-Eth yl-6-a za u r a cil (16). To a solution of freshly prepared
sodium ethoxide [prepared from the reaction of sodium (3.00
g, 130.4 mmol) and ethanol (90 mL)] was added a suspension
of 14 (6.86 g, 43.38 mmol) in ethylene glycol (100 mL). The
reaction was refluxed at 100 °C for 48 h and then concentrated
in vacuo (high vacuum pump, 2.0 mmHg). The residue was
dissolved in the minimal amount of hot water and acidified to
pH 2 by the addition of concentrated hydrochloric acid. The
solution was left in a refrigerator (4 °C) for 12 h, and the
resulting white precipitate was filtered and dried in a vacuum
oven at 70 °C for 12 h to give 2.3 g (38%) of 16 as a white
crystalline solid: mp: 145-146 °C (lit. mp: 145-147 °C); 1H
NMR (DMSO-d6): δ 12.03 (s, 1, NH), 11.56 (s, 1, NH), 2.46 (q,
2, J ) 7.4 Hz, CH2) 1.06 (t, 3, J ) 7.4 Hz, CH3). 13C NMR
(DMSO-d6): δ 157.50 (CdO, C-4), 149.88 (Cq, C-5), 146.51
(CdO, C-5), 22.84 (CH2), 10.62 (CH3).
5-P r op yl-6-a za u r a cil (17). To a solution of freshly pre-
pared sodium ethoxide [prepared from the reaction of sodium
(4.6 g, 197.5 mmol) and ethanol (100 mL)] was added a
suspension of 15 (11.4 g, 65.83 mmol) in ethylene glycol (200
mL). The reaction was refluxed at 100 °C for 72 h and then
concentrated in vacuo (high vacuum pump, 2.0 mmHg). The
residue was dissolved in the minimal amount of hot water and
acidified to pH 2 by the addition of concentrated hydrochloric
acid. The solution was left in a refrigerator for 12 h, and the
resulting white precipitate was filtered and dried in a vacuum
oven at 70 °C for 12 h to give 2.31 g (23%) (as determined
from NMR of the mixture of product:starting material 5:1) of
17 as a white solid. NMR describes the product (17) signals
only: 1H NMR (DMSO-d6): δ 12.04 (s, 1, NH), 11.87 (s, 1, NH),
2.41 (t, 2, J ) 7.5 Hz, CH2), 1.55 (m, 2, J ) 7.5 Hz, J ) 7.4 Hz,
CH2), 0.90 (t, 3, J ) 7.4 Hz, CH3). 13C NMR (DMSO-d6): δ
157.57 (CdO, C-4), 149.86 (Cq, C-5), 145.51 (CdO, C-2), 31.33
(CH2), 19.48 (CH2) 13.92 (CH3).
Gen er a l P r oced u r e for th e Syn th esis of 1-(2-Deoxy-4-
th io-3,5-di-O-ben zyl-r/â-D-er yth r o-pen tofu r an osyl)-(6-aza-
5-a lk yl-u r a cil) (19, 20, a n d 21). To a suspension of 6-aza-
5-alkyl-uracil (10, 16, or 17) (30 mmol) in anhydrous acetonitrile
(30 mL) was added N,O-bis(trimethylsilyl)acetamide (60 mmol),
and the reaction was stirred under nitrogen at room temper-
ature for 1 h. To the now clear solution was added crushed 4
Å activated molecular sieves (2.5 g) followed by a solution of
18 (36 mmol) in anhydrous acetonitrile (60 mL). After 10 min,
a solution of either iodine monochloride (36 mmol) or N-
iodosuccinimide (36 mmol) in anhydrous acetonitrile (35 mL)
(heat required to dissolve the N-iodosuccinimide) was added,
and the resulting brown solution was stirred under nitrogen
at room temperature for 24 h. The molecular sieves were
removed by filtration, and the filtrate was concentrated in
vacuo to give a brown syrup, which was dissolved in dichloro-
methane (300 mL) and washed with a saturated aqueous
sodium thiosulfate solution (150 mL). The organic layer was
dried (MgSO4) and concentrated in vacuo to give a red/brown
syrup. Purification by achieved by flash column chromatog-
raphy.
much improved toxicity profile compared with 4′S-T.
4′S-T displayed significant cytotoxicity to rapidly pro-
liferating cell populations (L1210, CCRF-CEM and
human epidermoid carcinoma #2).18 In contrast 5â
displayed cytotoxicity data comparable with ganciclovir
(5â: MCC > 200 µM, CC50 166 µM; ganciclovir: MCC
and CC50 > 150 µM) in human embryonic lung (Table
5), and cytotoxicity comparable with acyclovir and
brivudin (5â: MCC > 200 µg/mL; acyclovir/brivudin:
MCC > 400 µg/mL) in E6SM cell cultures (Table 6).
Compound 5â showed an interesting activity against
herpes simplex virus type 1 and type 2 and varicella-
zoster virus. Interestingly, it only lost antiviral efficacy
by 5- to 15-fold against thymidine kinase-deficient
HSV-1 and VZV strains. These observations may point
to a partial dependence on virus-encoded TK for its
eventual antiviral activity. These findings may also
indicate that this compound may be inhibitory in its
parental nucleoside form or use an alternative metabolic
pathway for conversion to its antivirally active deriva-
tive. When evaluated for its inhibitory activity against
phosphorylation of radiolabeled thymidine by purified
cytosolic TK-1, mitochondrial TK-2, HSV-1 TK, and VZV
TK, 5â was not inhibitory toward TK-1, showed a
marginal inhibitory effect toward TK-2 (IC50: ∼g500
µM), but was endowed with a pronounced inhibitory
potential toward HSV-1 TK (IC50: 5.0 ( 0.94 µM) and
VZV TK (IC50: 33 ( 5.4 µM). These data suggest that
the compound is most likely converted to its 5′-mono-
phosphate by HSV-1 TK and VZV TK, but not cytosolic
TK-1. Further studies are required to elucidate the
metabolic characteristics of 5â and to pinpoint its
molecular target(s) against HSV and VZV.
Exp er im en ta l Section
Gen er a l P r oced u r es. 1H and 13C NMR spectra were
recorded with a Brucker Avance DPX300 spectrometer operat-
ing at 300 and 75 MHz, with Me4Si as internal standard. FAB
Mass spectra were determined by the EPSRC mass spectrom-
etry center (Swansea, UK). Microanalyses were determined
by Medac Ltd (Surrey, UK). Flash column chromatography
was performed with silica gel 60 (230-400mesh) (Merck) and
TLC was carried out on precoated silica plates (kiesel gel 60
F
254, BDH). Compounds were visualized by illumination under
UV light (254 nm) or by the use of vanillin stain followed by
charring on a hotplate. Melting points were determined on an
electrothermal instrument and are uncorrected. All solvents
were dried prior to use as described by the handbook Purifica-
tion of Laboratory Chemicals22 and stored over 4 Å molecular
sieves, under nitrogen. Optical rotation was not undertaken
for the final compounds 5-9 owing to the limited amounts of
samples and the very limited additional information provided
by such a measurement.
Sem ica r ba zon e of 2-Ketobu tyr ic Acid (14). To a solution
of semicarbazide‚HCl 11 (10.92 g, 97.95 mmol) in water (60
mL) was added 2-ketobutyric acid 12 (10 g, 97.95 mmol) in
small portions. The solution was stirred at room temperature
for 1 h. The resulting white precipitate was filtered, washed
with cold water, and dried under vacuum at 70 °C for 12 h to
give 14.15 g (91%) of 14 as a white solid: mp: 180-182 °C
(lit. mp: 188-189 °C); 1H NMR (DMSO-d6): δ 10.10 (s, 1, NH),
7.35 (bs, 1, NH2), 6.70 (bs, 1, NH2), 2.51 (q, 2, J ) 7.5 Hz, CH2)
0.90 (t, 3, J ) 7.5 Hz, CH3). 13C NMR (DMSO-d6): δ 164.95
(CdO, acid), 156.79 (CdO, amide), 140.86 (Cq), 18.02 (CH2),
10.25 (CH3).
1-(2-Deoxy-4-th io-3,5-d i-O-ben zyl-r/â-D-er yth r o-p en to-
fu r a n osyl)-(6-a za -th ym in e) (19). Purification by flash col-
umn chromatography (petroleum ether-ethyl acetate 3:2 v/v)
gave 13.54 g (78%) of the product 19 as a yellow hydroscopic
solid, R and â mixture of approximately 1:1: Rf 0.18 (petroleum
ether-ethyl acetate 3:2); 1H NMR (CDCl3) mixture of ano-
mers: δ 10.17 (bs, 1, NH R and â), 7.38 (m, 10, CH Ar R and
Sem ica r ba zon e of 2-Ketova ler ic Acid (15). To a solution
of semicarbazide‚HCl 11 (8.10 g, 72.63 mmol) in water (100
mL) was added 2-oxopentanoic acid sodium salt 13 (10 g, 72.41

5488 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Maslen et al.
â), 6.49 (dd, 1/2, J 1′,2′ ) 7.1 Hz, J 1′,2′ ) 5.7 Hz, H-1′ R/â), 6.34
(ψt, 1/2, J 1′,2′ ) 7.7 and 7.5 Hz, H-1′ R/â), 4.62 (m, 4, CH2 Bn
R and â), 4.55 (m, 1/2, H-3′ â), 4.02 (m, 1, H-5′ R and â), 3.89
(dd, 1/2, J ) 9.7 Hz, J ) 3.6 Hz, H-3′ R), 3.79 (m, 1, H-5′ R
and â), 3.62 (m, 1, H-4′ R and â), 2.65 (m, 1 1/2, H-2′ R and â),
2.46 (m, 1/2, H-2′ R/â), 2.32 (s, 1 1/2, CH3 R/â), 2.24 (s, 1 1/2,
CH3 R/â). 13C NMR (CDCl3): δ 156.69, 156.65 (2 × CdO, C-4),
149.04, 148.92 (2 × Cq, C-5), 145.32, 145.00 (2 × CdO, C-2),
138.39, 138.36, 138.30, 138.17 (4 × Cq, Ar), 128.96, 128.90,
128.87, 128.42, 128.27, 128.23, 128.17, 128.10 (20 × CH, Ar),
82.98, 82.31 (2 × CH, C-3′), 73.70, 73.52, 73.14, 72.95 (8 ×
CH2, Bn), 72.04, 70.96 (2 × CH2, C-5′), 63.37, 59.94 (2 × CH,
C-1′), 53.18, 52.92 (2 × CH, C-4′), 38.68, 37.73 (2 × CH2, C-2′),
17.14, 17.02 (2 × CH3). HRMS (ES+) (anomeric mixture) calcd
for C23H26N3O4S (M + H)+ 440.1644, found, 440.1640.
raphy (chloroform-methanol 9: 1 v/v) gave the product 5 as
the anomers R (pale yellow solid) and â (off white solid).
Yield: 5R 3.45 g (43%) and 5â 1.56 g (20%); mp 5R 153-155
°C and 5â 153 °C (decomposes); Rf 5R 0.22 and 5â 0.18
(chloroform/methanol 9:1); R-anomer 5R : 1H NMR (CD3OD):
δ 6.34 (dd, 1, J 1′,2′ ) 8.1 Hz, J 1′,2′ ) 5.9 Hz, H-1′), 4.22 (m, 1,
H-3′), 3.80 (dd, 1, J 5′,5′ ) 10.1 Hz, J 5′,4′ ) 4.3 Hz, H-5′), 3.60
(m, 2, H-5′ and H-4′), 2.65 (m, 1, H-2′), 2.51 (m, 1, H-2′), 2.24
(s, 3, CH3). 13C NMR (CD3OD): δ 158.63 (CdO, C-4), 150.62
(Cq, C-5), 146.67 (CdO, C-2), 76.73 (CH, C-3′), 65.27 (CH2,
C-5′), 62.16 (CH, C-1′), 59.83 (CH, C-4′), 41.46 (CH2, C-2′),
16.98 (CH3). UV (MeOH) λmax ) 296 nm (ꢀ 1770), 290 nm (ꢀ
1530). HRMS (CI) Calcd for C9H17N4O4S (M + H)+ 260.0705,
found 260.0710. â-anomer 5â: 1H NMR (CD3OD): δ 6.37 (dd,
1, J 1′,2′ ) 7.3 Hz, J 1′,2′ ) 5.1 Hz, H-1′), 4.71 (m, 1, H-3′), 3.89
(dd, 1, J 5′,5′ ) 11.3 Hz, J 5′,4′ ) 6.8 Hz, H-5′), 3.68 (dd, 1, J 5,5
)
1-(2-Deoxy-4-th io-3,5-d i-O-ben zyl-r/â-D-er yth r o-p en to-
fu r a n osyl)-(5-eth yl-6-a za u r a cil) (20). Purification by flash
column chromatography (petroleum ether-ethyl acetate 7:3
v/v) gave 9.61 g (70%) of the product 20 as a thick syrup, R
and â mixture of approximately 1:1: Rf 0.26 (petroleum ether-
ethyl acetate 3:2); 1H NMR (CDCl3) mixture of anomers: δ
9.74 (s, 1, NH R and â), 7.37 (m, 10, CH Ar R and â), 6.50 (dd,
11.2 Hz, J 5,4 ) 6.9 Hz, H-5′) 3.39 (m, 1, H-4′), 2.70 (m, 1, H-2′),
2.35 (m, 1, H-2′), 2.64 (s, 3, CH3). 13C NMR (CD3OD): δ 158.70
(CdO, C-4), 150.77 (Cq, C-5), 146.04 (CdO, C-2), 76.74 (CH,
C-3′), 66.22 (CH2, C-5′), 63.60 (CH, C-1′), 59.53 (CH, C-4′),
41.72 (CH2, C-2′), 17.00 (CH3). UV (MeOH) λmax ) 296 nm (ꢀ
1370), 291 nm (ꢀ 1310). Anal. (C9H13N3O4S) C, H, N, S.
1/2, J 1′,2′ ) 7.3 Hz, J 1′,2′ ) 5.4 Hz, H-1′ R/â), 6.33 (t, 1/2, J 1′,2′
)
1-(2-Deoxy-4-th io-r- a n d â-D-er yth r o-p en tofu r a n osyl)-
(5-eth yl-6-a za u r a cil) (6). Purification by flash column chro-
matography (chloroform-methanol 95:5 v/v) gave the product
6 as the anomers, R (white solid) and â (white solid). Yield:
6R 2.31 g (40%) and 6â 1.01 g (17%); mp 6R 68-70 °C and 6â
189-191 °C; Rf 6R 0.42 and 5â 0.35 (chloroform/methanol 4:1);
R-anomer 6R : 1H NMR (DMSO-d6): δ 12.15 (s, 1, NH), 6.13
(t, 1, J ) 7.7 Hz, H-1′), 5.30 (d, 1, J ) 6.6 Hz, OH), 4.87 (ψt,
1, J ) 5.2 Hz and J ) 4.9 Hz, OH), 3.92 (m, 1, H-3′), 3.82 (m,
1, H-5′), 3.45 (m, 1, H-5′), 3.33 (m, 1, H-4′), 2.49 (m, 4, CH2
and H-2′), 1.14 (t, 3, J ) 7.4 Hz, CH3). 13C NMR (DMSO-d6):
δ 156.60 (CdO, C-4), 148.69 (Cq, C-5), 147.71 (CdO, C-2),
72.25 (CH, C-3′), 63.52 (CH2, C-5′), 58.84 (CH, C-1′), 57.88 (CH,
C-4′), 39.81 (CH2, C-2′), 23.24 (CH2), 10.40 (CH3). UV (MeOH)
λmax ) 295 nm (ꢀ 1820), 291 nm (ꢀ 1850). HRMS (CI) Calcd for
7.6 Hz, H-1′ R/â), 4.60 (m, 4 1/2, CH2 Bn R and â, H-3′ â), 4.01
(m, 1, H-5′ R and â), 3.90 (m, 1/2, H-3′ R), 3.79 (m, 1, H-5′ R
and â), 3.61 (m, 1, H-4′ R and â), 2.66 (m, 3 1/2, CH2 R and â,
H-2′ R/â), 2.48 (m, 1/2, H-2′ R/â), 1.29 (t, 1 1/2, J ) 7.4 Hz CH3
R/â), 1.17 (t, 1 1/2, J ) 7.4 Hz CH3 R/â). 13C NMR (CDCl3): δ
156.27 (2 × CdO, C-4), 148.87, 148.76, 148.65, 145.61 (2 ×
CdO, C-2) and (2 × Cq, C-5), 138.35, 138.29, 138.17, (4 × Cq,
Ar), 128.93, 128.90, 128.85, 128.41, 128.25, 128.22, 128.16,
128.07 (20 × CH, Ar), 83.10, 82.32 (2 × CH, C-3′), 73.68, 73.56,
73.18, 73.01 (4 × CH2, Bn), 72.11, 7088 (2 × CH2, C-5′), 63.25,
59.98 (2 × CH, C-1′), 53.16, 52.87 (2 × CH, C-4′), 38.63, 37.91
(2 × CH2, C-2′), 23.99, 23.85 (2 × CH2), 10.73, 10.57 (2 × CH3).
Anal. (C24H27N3O4S) C, H, N, S.
1-(2-Deoxy-4-th io-3,5-d i-O-ben zyl-r/â-D-er yth r o-p en to-
fu r a n osyl)-(5-p r op yl-6-a za u r a cil) (21). Purification by flash
column chromatography (petroleum ether-ethyl acetate 7:3
v/v) gave 4.37 g (76%) of the product 21 as a thick orange
syrup, R and â mixture of approximately 1:1: Rf 0.30 (petro-
leum ether-ethyl acetate 3:2); 1H NMR (CDCl3) mixture of
anomers: δ 9.69 (s, 1, NH R and â), 7.34 (m, 10, CH Ar R and
â), 6.48 (dd, 1/2, J 1′,2′ ) 7.1 Hz, J 1′,2′ ) 5.5 Hz, H-1′ R/â), 6.30
(t, 1/2, J ) 7.6 Hz, H-1′ R/â), 4.58 (m, 4, CH2 Bn R and â), 4.53
(m, 1/2, H-3′ R/â), 3.98 (m, 1, H-5′ R and â), 3.88 (m, 1/2, H-3′
R/â), 3.77 (m, 1, H-5′ R and â), 3.60 (m, 1, H-4′ R and â), 2.64
(m, 3 1/2, CH2 R and â, H-2′ R/â), 2.46 (m, 1/2, H-2′ R/â), 1.68
(m, 2, CH2 R and â), 1.00 (m, 3, CH3 R and â). 13C NMR
(CDCl3): δ 156.37, 156.33 (2 × CdO, C-4), 148.80, 148.69 (2
× Cq, C-5), 148.00, 147.66 (2 × CdO, C-4), 138.36, 138.31,
138.19, (4 × Cq, Ar), 128.94, 128.89, 128.85, 128.41, 128.25,
128.22, 128.15, 128.05 (20 × CH, Ar), 83.04, 82.32 (2 × CH,
C-3′), 73.66, 73.55, 73.20, 73.05 (4 × CH2, Bn), 72.06, 70.86 (2
× CH2, C-5′), 63.22, 59.89 (2 × CH, C-1′), 53.16, 52.84 (2 ×
CH, C-4′), 38.65, 37.91 (2 × CH2, C-2′), 32.26, 32.14 (2 × CH2),
19.90, 19.67 (2 × CH2), 14.15, 14.13 (2 × CH3). Anal.
(C25H29N3O4S) C, H, N, S.
Gen er a l P r oced u r e for th e Syn th esis of 1-(2-Deoxy-4-
th io-r- a n d â-D-er yth r o-p en tofu r a n osyl)-(6-a za -5-a lk yl-
u r a cil) (5, 6, a n d 7). To a cooled (-80 °C) solution of boron
trichloride (1 M in dichloromethane, 25 mL, 25 mmol) in
anhydrous dichloromethane (25 mL) was added dropwise a
solution of 19, 20, or 21 (10 mmol) in anhydrous dichloro-
methane (25 mL). The reaction was stirred at -80 °C for 4 h,
and then the reaction was quenched by adding a 1:1 mixture
of methanol/dichloromethane (50 mL) dropwise at -80 °C and
allowed to warm to room temperature. The reaction was
concentrated in vacuo to give a brown solution, which was
coevaporated with methanol (3 × 15 mL) to give a brown
syrup. Purification was achieved by flash column chromatog-
raphy.
C
10H15N3O4S (M + H)+ 274.0861, found 274.0863. â-anomer
6â: 1H NMR (DMSO-d6): δ 12.13 (s, 1, NH), 6.19 (ψt, 1, J )
6.8 Hz and J ) 5.5 Hz, H-1′), 5.21 (d, 1, J ) 4.0 Hz, OH), 4.96
(bs, 1, OH), 4.49 (m, 1, H-3′), 3.70 (ψt, 1, J ) 8.4 Hz and J )
7.7 Hz, H-5′), 3.38 (m, 1, H-5′), 3.21 (m, 1, H-4′), 2.50 (m, 3,
CH2 and H-2′), 2.17 (m, 1, H-2′), 1.12 (t, 3, J ) 7.3 Hz, CH3).
13C NMR (DMSO-d6): δ 156.62 (CdO, C-4), 148.72 (Cq, C-5),
147.29 (CdO, C-2), 74.44 (CH, C-3′), 64.92 (CH2, C-5′), 61.58
(CH, C-1′), 58.69 (CH, C-4′), 40.29 (CH2, C-2′), 23.11 (CH2),
10.21 (CH3). UV (MeOH) λmax ) 295 nm (ꢀ 1710), 291 nm (ꢀ
1710). HRMS (CI) Calcd for C10H15N3O4S (M + H)+ 274.0861,
found 274.0865.
1-(2-Deoxy-4-th io-r- a n d â-D-er yth r o-p en tofu r a n osyl)-
(5-p r op yl-6-a za u r a cil) (7). Purification by flash column
chromatography (chloroform-methanol 96:4 v/v) gave the
product 7 as the anomers, R (white solid) and â (white solid).
Yield: 7R 1.07 g (40%) and 7â 0.84 g (31%); mp 7R 125-126
°C and 7â 134-135 °C; Rf 7R 0.47 and 7â 0.42 (chloroform/
methanol 4:1); R-anomer 7R: 1H NMR (CD3OD): δ 6.13 (dd,
1, J 1′,2′ ) 8.2 Hz, J 1′,2′ ) 5.9 Hz, H-1′), 4.30 (m, 1, H-5′), 3.89
(m, 1, H-5′), 3.68 (m, 2, H-5′ and H-4′), 2.73 (m, 3, CH2 and
H-2′), 2.61 (m, 1, H-2′), 1.86 (m, 2, J ) 7.4 Hz, CH2), 1.11 (t, 3,
J ) 7.4 Hz, CH3). 13C NMR (CD3OD): δ 158.39 (CdO, C-4),
150.49 (Cq, C-5), 149.27 (CdO, C-2), 76.73 (CH, C-3′), 65.24
(CH2, C-5′), 62.10 (CH, C-1′), 59.81 (CH, C-4′), 41.46 (CH2,
C-2′), 33.22 (CH2), 21.01 (CH2), 14.43 (CH3). UV (MeOH) λmax
) 297 nm (ꢀ 2260), 291 nm (ꢀ 2030). Anal. (C11H17N3O4S) C,
H, N, S. â-anomer 7â: 1H NMR (CD3OD): δ 6.30 (dd, 1, J 1′,2′
) 7.3 Hz, J 1′,2′ ) 4.7 Hz, H-1′), 4.64 (m, 1, H-3′), 3.82 (m, 1,
J 5′,5′ ) 11.2 Hz, H-5′), 3.59 (m, 1, J 5′,5′ ) 11.2 Hz, H-5′), 3.34
(m, 1, H-4′), 2.57 (m, 3, CH2 and H-2′), 2.29 (m, 1, H-2′), 1.69
(m, 2, CH2), 0.99 (t, 3, J ) 7.3 Hz, CH3). 13C NMR (CD3OD):
δ 158.48 (CdO, C-4), 150.61 (Cq, C-5), 148.57 (CdO, C-2),
76.76 (CH, C-3′), 66.33 (CH2, C-5′), 63.40 (CH, C-1′), 59.44 (CH,
C-4′), 41.71 (CH2, C-2′), 33.11 (CH2), 20.82 (CH2), 14.47 (CH3).
UV (MeOH) λmax ) 296 nm (ꢀ 2180), 291 nm (ꢀ 1980). Anal.
(C11H17N3O4S) C, H, N, S.
1-(2-Deoxy-4-th io-r- a n d â-D-er yth r o-p en tofu r a n osyl)-
(6-a za th ym in e) (5). Purification by flash column chromatog-

6-Azapyrimidine-2′-deoxy-4′-thionucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5489
Gen er a l P r oced u r e for th e Syn th esis of 1-(2-Deoxy-2-
iod o-4-th io-r/â-D-er yth r o-p en tofu r a n osyl)-(6-a za -5-a lk yl-
u r a cil) (23, 24, a n d 25). To a 0 °C solution of 18 (10 mmol)
in anhydrous dichloromethane (55 mL) was added 2,6-di-tert-
butyl-4-methylpyridine (15 mmol) followed by the dropwise
addition of iodine monochloride (12 mmol) in anhydrous
dichloromethane (13 mL) over 10 min under nitrogen. The
reaction was then stirred at 0 °C for 5 h. A freshly prepared
solution of bisilylated 6-azapyrimidine [prepared by the reac-
tion of 6-azapyrimidine 10, 16, or 17 (15 mmol) in dichloro-
methane (30 mL) and N,O-bis(trimethylsilyl)acetamide (30
mmol)] was added dropwise followed by a further addition of
iodine monochloride (12 mmol). The reaction was warmed to
room temperature and stirred under nitrogen for 16 h. The
reaction was quenched by the addition of saturated aqueous
sodium thiosulfate (50 mL) and the product extracted by
washing with dichloromethane (3 × 25 mL). The organic layer
was dried (MgSO4) and concentrated in vacuo to give an orange
syrup. Purification was achieved by flash column chromatog-
raphy.
138.07, 137.86, 137.49 (4 × Cq, Ar), 128.94, 128.91, 128.84,
128.67, 128.45, 128.36, 128.07 (20 × CH, Ar), 87.26, 83.04 (2
× CH, C-3′), 75.19, 73.84, 73.66, 72.70, (4 × CH2, Bn), 72.55,
70.48 (2 × CH2, C-5′), 69.37, 64.22 (2 × CH, C-1′), 50.41, 50.21
(2 × CH, C-4′), 32.24, 32.12 (2 × CH2), 29.17, 29.10 (2 × CH,
C-2′), 19.95, 19.61 (2 × CH2), 14.09 (2 × CH3). HRMS (ES+)
Calcd for (M + NH4)+ 611.1189, found: 611.1187.
1-(2-Deoxy-3,5-di-O-acetyl-4-th io-â-D-er yth r o-pen tofu r an -
osyl)-(6-a za th ym in e) (26). To a solution of 5â (1.5 g, 5.79
mmol) in anhydrous pyridine (20 mL) was added acetic
anhydride (2.1 mL, 23.14 mmol). The reaction was stirred at
0 °C for 15 min and then at room temperature for 16 h. The
reaction was quenched with N,N-dimethylethylenediamine (4
mL, 37.58 mmol) in dichloromethane (50 mL) while cooling
the reaction flask in ice. The organic layer was then washed
with 2 M aqueous HCl (30 mL), saturated aqueous sodium
bicarbonate (30 mL), and water (30 mL), dried (MgSO4), and
concentrated in vacuo to give a yellow liquid, which was
azeotroped with toluene to give a yellow syrup. Purification
by flash column chromatography (chloroform-methanol 19:1
v/v) gave 1.50 g (75%) of 26 as a pale yellow syrup: Rf 0.69
1-(2-Deoxy-2-iod o-4-th io-3, 5-d i-O-ben zyl-r/â-D-er yth r o-
p en tofu r a n osyl)-(6-a za th ym in e) (23). Purification by flash
column chromatography (petroleum ether-ethyl acetate 7:3
v/v) gave 3.48 g (70%) of 23 as a thick orange syrup, R and â
mixture of approximately 1:4: Rf 0.32 (petroleum ether-ethyl
acetate 3:2); Anomeric mixture, R:â, 1:4, Major anomer (â)
signals described 1H NMR (CDCl3) δ 9.92 (s, 4/5, NH â), 7.52
(m, 10, CH Ar â), 6.61 (d, 4/5, J 1′,2′ ) 8.4 Hz, H-1′ â), 5.01 (dd,
4/5, J 2′,1′ ) 8.4 Hz, J 2′,3′ ) 3.9 Hz, H-2′ â), 4.83 (s, 8/5, CH2Ph
â), 4.69 (s, 8/5, CH2Ph â), 4.25 (ψt, 4/5, J ) 3.5 and 2.8 Hz,
H-3′ â), 3.94 (m, 4/5, J 4′,5′ ) 7.0 Hz, J 4′,3′ ) 2.5 Hz, H-4′ â),
3.75 (d, 8/5, J 5′,5′ ) 7.0 Hz, J 5′,4′ ) 2.5 Hz, H-5′ â), 2.36 (s, 12/5,
CH3 â). Major anomer (â) signals described 13C NMR (CDCl3)
δ 156.31 (CdO, C-4), 148.81 (Cq, C-5), 145.66 (CdO, C-2),
138.12, 137.48 (2 × Cq, Ar), 128.94, 128.85, 128.73, 128.48,
128.35, 128.08 (10 × CH, Ar), 82.95 (CH, C-3′), 73.65, 72.73,
(2 × CH2, Bn), 72.44 (CH2, C-5′), 69.35 (CH, C-1′), 50.48 CH,
C-4′), 29.44 (CH2, C-2′), 17.12 (CH3). HRMS (ES+) Calcd for
(M + H)+ 566.0611, found 566.0594.
1
(chloroform/methanol 4:1); H NMR (CDCl3): δ 10.23 (bs, 1,
NH), 6.54 (ψt, 1, J ) 7.0 Hz, J ) 6.4 Hz, H-1′), 5.63 (m, 1,
H-3′), 4.37 (dd, 1, J 5′,5′ ) 11.4 Hz, J 5′,4′ ) 8.6 Hz, H-5′), 4.26
(dd, 1, J 5′,5′ ) 11.5 Hz, J 5′,4′ ) 6.6 Hz, H-5′), 3.70 (m, 1, H-4′),
2.82 (m, 1, H-2′), 2.48 (m, 1, H-2′), 2.20 (s, 3, CH3), 2.14 (s, 3,
CH3 Ac), 2.12 (s, 3, CH3 Ac). 13C NMR (CDCl3): δ 171.15 (CdO,
Ac), 170.85 (CdO, Ac), 156.54 (CdO, C-4), 149.07 (CdO, C-2),
145.82 (Cq, C-5), 77.95 (CH, C-3′), 65.87 (CH2, C-5′), 63.33 (CH,
C-1′), 52.15 (CH, C-4′), 38.34 (CH2, C-2′), 21.55 (CH3, Ac), 21.22
(CH3, Ac), 17.14 (CH3). HRMS (ES+) Calcd for (M + NH4)+
361.1182, found 361.1181.
1-(2-Deoxy-3,5-di-O-acetyl-4-th io-â-D-er yth r o-pen tofu r an -
osyl)-(4-th io-6-a za th ym in e) (27). To a solution of 26 (1.65
g, 4.81 mmol) in anhydrous toluene (20 mL) was added
Lawesson’s Reagent (2.33 g, 5.77 mmol), and the reaction was
refluxed under nitrogen for 3 h. The reaction was quenched
by coevaporation with methanol to give a red/brown syrup.
Purification by flash column chromatography (dichloro-
methane-ethyl acetate 94:6 v/v) gave 1.10 g (64%) of 27 as a
bright orange hydroscopic solid. A sample was prepared for
high-resolution mass spectrometry and testing by preparative
TLC. Rf 0.33 (dichloromethane/ethyl acetate 9:1); 1H NMR
(CDCl3): δ 10.90 (bs, 1, NH), 6.57 (ψt, 1, J ) 6.9 Hz, J ) 6.4
1-(2-Deoxy-2-iod o-4-th io-3,5-d i-O-ben zyl-r/â-D-er yth r o-
p en tofu r a n osyl)-(5-eth yl-6-a za u r a cil) (24). Purification by
flash column chromatography (petroleum ether-ethyl acetate
7:3 v/v) gave 2.9 g (67%) of 24 as a thick orange syrup, R and
â mixture of approximately 1:3: Rf 0.42 (petroleum ether-
ethyl acetate 3:2); Anomeric mixture, R:â, 1:4, Major anomer
(â) signals described 1H NMR (CDCl3) δ 9.65 (bs, 1, NH â),
7.33 (m, 10, CH Ar â), 6.47 (d, 3/4, J 1′,2′ ) 8.2 Hz, H-1′ â), 4.86
(dd, J 2′,1′ ) 8.2 Hz, J 2′,3′ ) 3.9 Hz, H-2′ â), 4.67 (s, 11/2, CH2Ph
Hz, H-1′), 5.71 (m, 1, H-3′), 4.45 (dd, 1, J 5′,5′ ) 11.3 Hz, J 5′,4′
)
8.6 Hz, H-5′), 4.33 (dd, 1, J 5′,5′ ) 11.5 Hz, J 5′,4′ ) 6.6 Hz, H-5′),
3.76 (m, 1, H-4′), 2.91 (m, 1, H-2′), 2.55 (m, 1, H-2′), 2.53 (s, 3,
CH3), 2.20 (s, 3, CH3 Ac), 2.19 (s, 3, CH3 Ac). 13C NMR
(CDCl3): δ 182.67 (CdS, C-4), 171.18 (CdO, Ac) 170.80 (CdO,
Ac), 148.78 (Cq, C-5), 146.05 (CdO, C-2), 78.01 (CH, C-3′),
65.85 (CH2, C-5′), 63.67 (CH, C-1′), 52.24 (CH, C-4′), 38.44
(CH2, C-2′), 21.57 (CH3, Ac), 21.25 (CH3, Ac), 20.83 (CH3).
HRMS (ES+) Calcd for (M + NH4)+ 377.0953, found 377.0949.
â), 4.10 (t, 3/4, J 3′,2′ ) J 3′,4′ ) 3.3 Hz, H-3′), 3.79 (m, 1, J 4′,5′
)
6.9 Hz, J 4′,3′ ) 2.6 Hz, H-4′â), 3.58 (d, 11/2, J 5′,4′ ) 7.0 Hz, H-5′
â), 2.63 (m, 11/2, CH2â), 1.13 (t, 21/4, J ) 7.3 Hz, CH3 â). 13C
NMR (CDCl3) δ 156.05 (2 × CdO, C-4), 149.95, 149.18 (2 ×
Cq, C-5), 148.97, 148.70 (2 × CdO, C-2), 138.07, 137.95, 137.49
(4 × Cq, Ar), 128.94, 128.90, 128.85, 128.66, 128.46, 128.36,
128.09 (20 × CH, Ar), 87.33, 83.07 (2 × CH, C-3′), 75.20, 73.84,
73.67, 72.73, (4 × CH2, Bn), 72.47, 70.49 (CH2, C-5′), 69.52,
64.34 (2 × CH, C-1′), 50.46, 50.22 (2 × CH, C-4′), 29.27, 29.17
(2 × CH2, C-2′), 24.04, 23.92 (2 × CH2) 10.72, 10.48 (2 × CH3).
HRMS (ES+) Calcd for (M + H)+ 580.0767, found: 580.0767.
1-(2-Deoxy-4-th io-â-D-er yth r o-p en tofu r a n osyl)-(4-th io-
6-a za th ym in e) (8). Compound 27 (1 g, 2.78 mmol) was
dissolved in methanolic ammonia (50 mL) at 0 °C and stirred
under nitrogen at 0 °C for 4 h. The reaction was warmed to
room temperature where it was stirred under nitrogen for a
further 4 h. The reaction was concentrated in vacuo to give
an orange foam. Purification by flash column chromatography
(dichloromethane-ethyl acetate 94:6 v/v) gave 0.5 g (66%) of
the product as a pale orange hydroscopic solid: Rf 0.57
1-(2-Deoxy-2-iod o-4-th io-3,5-d i-O-ben zyl-r/â-D-er yth r o-
p en tofu r a n osyl)-(5-p r op yl-6-a za u r a cil) (25). Purification
by flash column chromatography (petroleum ether-ethyl
acetate 7:3 v/v) gave 3.37 g (67%) of 25 as a thick orange syrup,
R and â mixture of approximately 1:3. Rf 0.43 (petroleum
ether-ethyl acetate 7:3); Anomeric mixture, R:â, 1:3, Major
anomer (â) signals described 1H NMR (CDCl3) δ 9.48 (s, 3/4,
NH â), 7.43 (m, 10, CH2Phâ), 6.56 (d, 3/4, J 1′,2′ ) 8.5 Hz, H-1′
â), 4.93 (dd, 3/4, J 2′,1′ ) 8.5 Hz, J 2′,3′ ) 3.9 Hz, H-2′â), 4.76 (s,
1
(chloroform/methanol 3:2); H NMR (CD3OD): δ 6.29 (dd, 1,
J 1′,2′ ) 7.3 Hz, J 1′,2′ ) 5.0 Hz, H-1′), 4.68 (m, 1, H-3′) 3.86 (dd,
1, J 5′,5′ ) 11.3 Hz, J 5′,4′ ) 6.7 Hz, H-5′), 3.65 (dd, 1, J 5′,5′ ) 11.3
Hz, J 5′,4′ ) 6.9 Hz, H-5′), 3.39 (m, 1, H-4′), 2.69 (m,1, H-2′),
2.38 (s, 3, CH3), 2.37 (m, 1, H-2′). 13C NMR (CD3OD): δ 185.39
(CdS, C-4), 148.95 (Cq, C-5), 147.750 (CdO, C-2), 76.78 (CH,
C-3′), 66.17 (CH2, C-5′), 63.85 (CH, C-1′), 59.60 (CH, C-4′),
41.77 (CH2, C-2′), 20.92 (CH3). UV (MeOH) λmax ) 297 nm (ꢀ
1730), 291 nm (ꢀ 1590). HRMS (ES+) Calcd for (M + NH4)+
293.0742, found 293.0738.
11/2, CH2Ph â), 4.61 (s, 11/2, CH2Ph â), 4.19 (ψt, 3/4, J 3′,2′
)
3.5 Hz, J 3′,4′ ) 2.7 Hz, H-3′ â), 3.88 (dd, 1, J 4′,5′ ) 7.1 Hz, J 4′,3′
) 2.4, H-4′ â), 3.66 (d, 11/2, J 5′,4′ ) 7.1 Hz, H-5′ â), 2.68 (m,
11/2, CH2 â), 1.71 (q, 11/2, CH2 â), 1.04 (t, 2 1/4, CH3 â). 13C
NMR (75 MHz, CDCl3) δ 160.28, 156.01 (2 × CdO, C-4),
148.89, 148.59, 148.20 (2 × Cq, C-5 and 2 × CdO, C-2), 138.68,
1-(2-Deoxy-4-th io-â-D-er yth r o-p en tofu r a n osyl)-(5-m eth -
yl-6-a za cytosin e) (9). Compound 27 (0.5 g, 1.82 mmol) was

5490 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Maslen et al.
dissolved in methanolic ammonia (30 mL) and heated at 30
°C in a sealed vessel for 72 h. The reaction was concentrated
in vacuo to give a pale orange solid. Trituration with acetone
and methanol gave 0.44 g (93%) of the product as a cream
solid: mp 68 °C (decomp); Rf 0.39 (chloroform-methanol 3:2);
1H NMR (DMSO-d6): δ 8.04 (bs, 1, NH2), 7.48 (bs, 1, NH2),
6.21 (ψt, 1, J ) 6.7 Hz, J ) 6.0 Hz, H-1′), 5.16 (bs, 1, OH) 4.96
(bs, 1, OH), 4.45 (m, 1, H-3′) 3.66 (dd, 1, J 5′,5′ ) 10.9 Hz, J 5′,4′
) 7.0 Hz, H-5′), 3.36 (dd, 1, J 5′,5′ ) 10.8 Hz, J 5′,4′ ) 7.3 Hz,
H-5′), 3.20 (m, 1, J 4′,5′ ) 7.0 Hz, H-4′), 2.46 (m, 1, H-2′), 2.16
(s, 3, CH3), 2.11 (m, 1, H-2′). 13C NMR (DMSO-d6): δ 158.64
(CdO), C-2), 154.06 (Cq, C-4), 134.28 (Cq, C-5), 74.40 (CH,
C-3′), 64.98 (CH2, C-5′), 62.06 (CH, C-1′), 58.42 (CH, C-4′),
40.37 (CH2, C-2′), 17.89 (CH3). UV (MeOH) λmax ) 296 nm (ꢀ
2240), 291 nm (ꢀ 2130). HRMS (ES+) Calcd for (M + Na)+
281.0684, found 281.0687.
X-r a y Str u ctu r e Deter m in a tion . A single-crystal suitable
for X-ray diffraction was grown by slow evaporation in a
narrow-necked tube using a saturated solution of 5â in
deuterated methanol. The X-ray measurements were made on
a crystal of approximate dimensions 0.20 × 0.20 × 0.10 mm.
Intensity data were obtained using graphite monochromated
Mo KR radiation (λ ) 0.71073 Å) at 150 K and a Bruker-
Nonius KappaCCD area detector diffractometer. φ and ω scans
were employed to fill the Ewald sphere using COLLECT
software.23 Unit cell refinement and data reduction were
undertaken using DENZO and SCALEPACK software.24,25
Compound 5â (C9H13N3O4S), FW ) 259.28, crystallized in
the orthorhombic space group P212121, with a ) 7.7150(10), b
) 8.3080(10), c ) 17.9590(10) Å, V ) 1151.1(2) Å3, Z ) 4, Dcalcd
) 1.496 mg/m3 and µ (Mo KR) ) 0.289 mm-1. The total data
recorded within θ range of 3.48 to 27.49° was 6187, which
merged to give 2572 unique data (Rint ) 0.034) of which 2431
were considered observed (Fo > 2σ(F)). A semiempirical scan
absorption correction was applied using SORTAV26 and values
of Tmin ) 0.9444 and Tmax ) 0.9717 were obtained. The
structure was solved using SHELX-S27 and refined using
SHELXL28 to give a final R1 value of 0.0300 for 206 param-
eters. The absolute structure parameter29 was 0.01(6). The
non-hydrogen atoms were refined anisotropically. H atoms
were found using the difference map and their positions refined
isotropically.
trays were inoculated with 100 CCID50 of virus, 1 CCID50 being
the virus dose required to infect 50% of the cell cultures. After
a 1 h virus adsorption period, residual virus was removed, and
the cell cultures were incubated in the presence of varying
concentrations (400, 200, 100, ... µg/mL) of the test compounds.
Viral cytopathicity was recorded as soon as it reached comple-
tion in the control virus-infected cell cultures.
The following viruses were included in the study: herpes
simplex virus type 1 (HSV-1, strain KOS), HSV-2 (strain G),
a thymidine kinase (TK)-deficient HSV-1 strain (HSV-1/TK-
ACVr), vaccinia virus and vesicular stomatitis virus (VSV) in
E6SM cell cultures, cytomegalovirus (strain AD169 and Davis),
varicella-zoster virus (strains YS and OKA) and TK-deficient
VZV (strains 07/1 and YS/R) in HEL cell cultures, vesicular
stomatitis virus, Coxsackie virus B4 and respiratory syncytial
virus (RSV) in HeLa cell cultures, and parainfluenza-3 virus,
reovirus-1, Sindbis virus, Coxsackie virus B4, and Punta Toro
virus in Vero cell cultures.
Ack n ow led gm en t. We would like to acknowledge
the EPSRC Mass Spectrometry Centre, Swansea, UK
for mass spectroscopy data, and the Geconcerteerde
Onderzoeksacties - Vlaanderen (Krediet no. 00/12).
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data for compound 5â. Tables of calculated H-bonded
assemblies for 5â and 4′ S-T using PLATON software and a
figure for comparison of H-bonded assemblies of 5â and 4′ S-T.
A table of elemental analyses. This material is available free
of charge via the Internet at http://pubs.acs.org.
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