Novel 3'-C/N-substituted 2',3'-β-D-dideoxynucleosides as potential chemotherapeutic agents. 1. Thymidine derivatives: Synthesis, structure, and broad spectrum antiviral properties

Article abstract of DOI:10.1021/jm960500w

A synthetic scheme for the 3'-oxime derivatives 3E, 5E, 5Z, 7E and 7Z of 1-(2,3-dideoxy-β-D-glycero-pentofuranosyl)thymine and for 1-(2,3-dideoxy-3- nitro-β-D-erythro-pentofuranosyl)-thymine (10) has been developed starting from appropriately 5'-protected

Full text of DOI:10.1021/jm960500w

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486
J . Med. Chem. 1997, 40, 486-494
Novel 3′-C/N-Su bstitu ted 2′,3′-â-D-Did eoxyn u cleosid es a s P oten tia l
Ch em oth er a p eu tic Agen ts. 1. Th ym id in e Der iva tives: Syn th esis, Str u ctu r e,
a n d Br oa d Sp ectr u m An tivir a l P r op er ties
Ivan I. Fedorov,*,† Ema M. Kazmina,† Galina V. Gurskaya,‡ Maxim V. J asko,‡ Valery E. Zavodnic,§
J an Balzarini, Erik De Clercq, Abdesslem Faraj,| J ean-Pierre Sommadossi,| J ean-Louis Imbach,# and
Gilles Gosselin*,#
Moscow Medical Sechenov Academy, 2-6 B. Pirogovskaya Str., 119881 Moscow, Russia, Engelhardt Institute of Molecular
Biology, 32 Vavilov Str., 117984 Moscow, Russia, Karpov Institute of Physical Chemistry, 10 Obukha Str.,
103064 Moscow, Russia, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
B-3000 Leuven, Belgium, The University of Alabama at Birmingham, Department of Pharmacology, G 019 Volker Hall,
1670 University Boulevard, Birmingham, Alabama 35294-0019, and Laboratoire de Chimie Bioorganique,
UMR CNRS-USTL 5625, Universite´ Montpellier II, Sciences et Techniques du Languedoc, 34095 Montpellier Ce´dex 5, France
Received J uly 9, 1996X
A synthetic scheme for the 3′-oxime derivatives 3E, 5E, 5Z, 7E and 7Z of 1-(2,3-dideoxy-â-D-
glycero-pentofuranosyl)thymine and for 1-(2,3-dideoxy-3-nitro-â-D-erythro-pentofuranosyl)-
thymine (10) has been developed starting from appropriately 5′-protected 3′-ketothymidine.
X-ray analysis showed that 3′-N-hydroxyimino 3E and 3′-N-methoxyimino 5Z derivatives have
close molecular conformations: anti about the N1-C1′ bond, and gauche+ about the C4′-C5′
exocyclic bond. Their sugar conformations are C1′-exo-O4′-endo and C1′-exo-C2′-endo,
respectively. The antiviral assays in cell cultures demonstrated that 3′-N-hydroxyimino 3E
and 3′-N-acetoxyimino 7E + 7Z derivatives are endowed with significant activity against human
immunodeficiency virus (HIV) with EC50 values ranging between 0.02 and 0.40 µg/mL for both
HIV-1 and HIV-2. The other compounds 5E + 5Z and 10 were at least 2 orders of magni-
tude less active. The 3′-N-hydroxyimino derivative 3E also shows promising activity against
hepatitis B virus (HBV) (EC50 ) 0.25 µg/mL) and against herpes simplex virus type 1 (HSV-1)
and HSV-2.
In tr od u ction
from concomitant inhibition of human DNA polymerase
R5 and â6 by the corresponding 5′-triphosphate. The
3′-N-alkylated derivatives of AMT with methyl or
ethyl groups are devoid of anti-HIV activity, but their
5′-triphosphates show more selective inhibitory proper-
ties than does the parent nucleotide to reverse tran-
scriptases (RT) of HIV and avian myeloblastosis virus
(AMV) and do not inhibit DNA biosynthesis catalyzed
by human DNA polymerases.6,7 Another example of an
anti-HIV nucleoside analogue bearing nitrogen at C3′
is 1-(2,3-dideoxy-3-nitro-â-D-erythro-pentofuranosyl)-
thymine (10).8,9 The main conformational parameters
of this molecule, obtained from the X-ray analysis, are
very similar to one of the crystallographically indepen-
dent forms of AZT found in its crystalline state.10
However, this compound shows moderate inhibition of
HIV replication in MT-4 cell culture,8 though its 5′-
triphosphate is a highly effective and selective inhibitor
of DNA biosynthesis catalyzed by RT of HIV and AMV.10
Therefore it was interesting to resynthesize 10 in order
to estimate its antiviral potency against a broad number
of viruses. These considerations also prompted us to
synthesize the N-hydroxyimino derivative 3E, as well
as the N-methoxyimino 5E + 5Z and N-acetoxyimino
7E + 7Z derivatives (Figure 1) in order to evaluate their
biological properties.
Modified nucleosides are still the main therapeutic
agents in the treatment of patients with acquired
immune deficiency syndrome (AIDS).1,2 The need to
search for new modified nucleoside analogues has
become especially clear since the appearance of HIV
strains resistant to 1-(2,3-dideoxy-3-azido-â-D-erythro-
pentofuranosyl)thymine (AZT), 2′,3′-dideoxycytidine
(ddC), 2′,3′-dideoxyinosine (ddI), and 1-(2,3-dideoxy-â-
D-glycero-pent-2-enofuranosyl)thymine (d4T), four drugs
currently approved for treatment of HIV-infected indi-
viduals.1,2
The significance of hydroxyl group replacement at C3′
in 2′-deoxynucleosides by different substituents contain-
ing nitrogen in order to obtain anti-HIV compounds has
been illustrated previously. Thus, the first potent
nucleoside analogue used as a commercial anti-HIV
drug, AZT, contains an azido group at C3′.3 1-(2,3-
Dideoxy-3-amino-â-D-erythro-pentofuranosyl)thymine
(AMT) is also active against HIV, but it did not find a
practical application due to strong cytotoxicity4 resulting
* Addresses for correspondence: Dr. G. Gosselin, Laboratoire de
Chimie Bioorganique, UMR CNRS-USTL 5625, case courrier 008,
Universite´ Montpellier II, Sciences et Techniques du Languedoc, Place
Euge`ne Bataillon, 34095 Montpellier Ce´dex 5, France. Tel: (33) 4 67-
14-38-55. Fax: (33)
4 67 04 20 29. E-mail: gosselin@crit.univ-
montp2.fr. Dr. I. I. Fedorov, Moscow Medical Sechenov Academy,
Department of Pharmaceutical Chemistry Advantages, 2-6 B.
Pirogovskaya Str., 119881 Moscow, Russia.
† Moscow Medical Sechenov Academy.
The additional rationale for the syntheses of the above
mentioned oximes came independently from the concept
that the flattened sugar conformation like realized in
the title oximes seems preferable for the design of
biologically active nucleoside analogues,11,12 since they
are a number of examples of nucleosides with a flattened
sugar moiety possessing high biological activity.11,14
‡ Engelhardt Institute of Molecular Biology.
§ Karpov Institute of Physical Chemistry.
Katholieke Universiteit Leuven.
| The University of Alabama at Birmingham.
#
Universite´ Montpellier II.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00500-6 CCC: $14.00 © 1997 American Chemical Society

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Potential Chemotherapeutic Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 487
Sch em e 1a
F igu r e 1. Structure of (E)-1-[2,3-dideoxy-3-(N-hydroxyimino)-
â-D-glycero- pentofuranosyl]thymine (3E) and of the (E,Z)-O-
methyl ethers (5E, 5Z) and acetyl esters (7E, 7Z).
To the best of our knowledge the oxime 3E and the
acetyl esters 7E + 7Z have not been previously synthe-
sized or studied as antiviral agents, although the 5′-O-
(tert-butyldimethylsilyl) derivative of 3E was recently
prepared for synthetical purposes by Tronchet and co-
workers.15 Also, the O-methyl ether 5 was recently
described by the same group,16 but no biological evalu-
ation has been reported for this compound.
a
Reaction conditions: (i) hydroxylamine hydrochloride or O-
methylhydroxylamine hydrochloride/pyridine; (ii) 80% aqueous
acetic acid; (iii) CH3C(O)Cl/pyridine.
Sch em e 2a
In addition, the 5′-protected oximes 2E + 2Z, reported
in the present work, appear to be convenient precursors
for the synthesis of the 3′-nitro derivative 108,9 which
is an interesting subject for antiviral studies. This
nucleoside analogue could be easily transformed to the
correspondent nitronic salt 118 with more flattened
sugar conformation.
The present paper is devoted to the synthesis of the
above mentioned new nucleoside analogues as well as
conformational studies performed using X-ray analysis
for compounds 3E (E isomer) and 5Z (Z isomer) in
comparison with 1010 and natural thymidine. The
antiviral properties of these analogues in various cell
cultures infected by a variety of viruses are also
reported.
a
Reaction conditions: (i) CF3CO3H/CH3CN; (ii) 80% aqueous
acetic acid; (iii) Na2CO3/H2O.
The mixture of the protected oximes 2E + 2Z was
successfully used to prepare, in only two steps and with
an improved overall yield, the previously reported 3′-
nitro derivative 108,9 (Scheme 2). Thus, oxidation of 2E
+ 2Z using a solution of pertrifluoroacetic acid in
acetonitrile in the presence of anhydrous Na2HPO4 and
CO(NH2)2 gave a mixture of the 5′-protected erythro-
and threo- 3′-nitro derivatives 8 and 9 in a ratio of 7:1.
Deprotection of this mixture with 80% aqueous acetic
acid afforded exclusively the erythro diastereomer 10
with the natural configuration of the nitro group. The
nucleoside analogue 10 was identical (1H NMR, mass
spectrum) to the compound previously reported.8,9 Fi-
nally, treatment of an aqueous solution of 10 with Na2-
CO3 afforded the quantitative formation of the corre-
sponding sodium salt of the nitronic acid 11.8
Resu lts
Ch em istr y. As an appropriate synthon for the
synthesis of the desired 3′-oximes derivatives 3E, 5E +
5Z, and 7E + 7Z, 5′-(monomethoxytrityl)-3′-ketothymi-
dine (1)17was chosen (Scheme 1). Compound 1 was
treated with a saturated solution of hydroxylamine
hydrochloride in pyridine or with O-methylhydroxy-
lamine hydrochloride to give, in quantitative yields, the
mixture of isomeric protected oximes 2E + 2Z and 4E
+ 4Z in a ratio of 3:2 and 3:1, respectively. These
mixtures of 5′-protected oximes were separated by
preparative TLC to afford the analytical samples of
individual E and Z isomers. Deprotection of the iso-
meric mixture of 2E + 2Z by 80% aqueous acetic acid
gave exclusively the E isomer 3E. A similar deprotec-
tion of compounds 4E + 4Z afforded the mixture 5E +
5Z in a ratio of 3:1; the compounds were also separated
by preparative TLC in order to isolate analytical samples
of the individual isomers. The acylation of the oxime
hydroxyls of the 2E + 2Z mixture by acetyl chloride in
pyridine followed by deprotection of the monomethoxy-
trityl group yielded an 7E + 7Z isomeric mixture in a
ratio of 3:2. This pair of 7E + 7Z isomers was separable
neither by chromatography on silica gel nor by reverse
phase chromatography.
P h ysicoch em ica l P r op er ties. The structure of all
the reported compounds was ascertained by H NMR,
1
UV, mass spectrometry, and elemental C, H, N analysis
for final compounds. Also, 13C NMR was recorded for
3E, 5E, 5Z, and 7E, and X-ray analysis was carried out
for the compounds 3E and 5Z.
The sugar portion of 1H NMR spectra of all title
oximes consists of two separated ABX systems of
protons (5′,5′′,4′ and 2′,2′′,1′). However, these two
systems are more complicated due to the long distance
W coupling constant between H2′ and H4′ ranging from
1 to 2 Hz. The assignment of 2′ and 2′′ protons was
done in accordance with the C. Altona rule18 and the
presence of long-distance coupling was taken into the
consideration during this assignment.
The assignment of the E and Z isomers was performed
on the basis of X-ray analysis for compound 3E (E
isomer) and 5Z (Z isomer). The NMR spectra of the
oximes 3E and 5Z have some characteristic features.

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488 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ta ble 1. Chemical Shifts in 1H NMR Spectra of Nucleoside Analoguesa
chemical shift, ppm
Fedorov et al.
compd
H-6 q
H-1′
H-4′ m
H-5′ dd
H-5′′ dd
H-2′′
H-2′
Me d
2Eb
2Zb
3E
7.62
7.76
7.57
7.58
7.74
7.55
7.71
7.60
7.73
7.58
7.73
7.51
7.63
7.61
7.72
7.20 pt
6.39 pt
6.31 pt
6.38 pt
6.39 dd
6.28 dd
6.27 dd
6.38 dd
6.48 dd
6.30 pt
6.34 dd
6.48 dd
6.22 dd
6.34 dd
6.27 pt
4.70
4.94
4.64
4.65
4.82
4.68
4.65
4.82
4.92
4.78
5.00
4.56
4.20
4.58
4.50
3.61
3.94
3.89
3.59
3.84
3.90
4.08
3.74
e
3.98
4.13
3.67
3.54
3.81
4.04
3.48
3.29
3.82
3.40
3.23
3.82
3.80
3.48
3.47
3.91
3.92
3.46
e
3.55 dd
3.23 dd
3.30 dd
3.44 dd
3.19 dd
3.28 ddd
3.08 dd
3.62 dd
3.39 dd
3.50 dd
3.25 dd
3.11 ddd
2.65 ddd
3.10 ddd
3.23 ddd
2.79 ddd
3.06 ddd
2.90 ddd
2.72 ddd
3.03 ddd
2.92 ddd
2.90 ddd
3.13 ddd
3.18 ddd
3.13 ddd
3.10 ddd
2.54 m
1.36
1.29
1.82
1.37
1.30
1.82
1.84
1.37
1.34
1.83
1.83
1.54
1.39
1.83
1.80
4Ec
4Zc
5Ec
5Zc
6Ed
6Zd
7Ed
7Zd
8f
9f
2.92 m
2.60 m
2.84 ddd
10f
11
3.83
3.84
a
Spectra were recorded in CDCl3 for compounds 2E, 2Z, 4E, 4Z, 6E, 6Z, 8, and 9 and in D2O for compounds 3E, 5E, 5Z, 7E, 7Z, 10,
b
and 11; the chemical shifts of the signals of the protecting group are not cited. N-OH: s, 9.01 and 8.69 for compounds 2E and 2Z,
respectively. c N-OMe: s, 3.97, 3.89, 3.86, and 3.82 for compounds 4E, 4Z, 5E and 5Z, respectively. N-OCOMe: s, 2.23, 1.94, 2.16, and
d
1.83 for compounds 6E, 6Z, 7E, and 7Z, respectively. e Signal is overlapped. f H3′, m, 5.22, 5.16 and 5.36 for compounds 8, 9, and 10,
respectively.
Ta ble 2. Coupling Constants in 1H NMR Spectra of Nucleoside
The three-dimensional structures of compounds 3E
Analogues
and 5Z were determined by X-ray analysis. Figure 2
J , Hz
shows the obtained conformations and the accepted
atom numbering for 3E, 5Z, (and 10),10 respectively. The
conformational parameters of the above mentioned
molecules are presented in Table 3, as well as those of
natural thymidine21 and of two crystallographically
independent forms of AZT.22
compd 1′,2′ 1′,2′′ 2′,2′′ 2′,4′ 4′,5′ 4′,5′′ 5′,5′′
γ+, %
2E
2Z
3E
4E
4Z
5Ea
5Z
6E
6Z
7E
7Z
8c
7.7
9.2
6.3
7.6
8.9
6.2
8.2
7.8
9.2
6.2
8.2
8.1
7.5
7.7
7.6
6.8
6.5
7.4
6.8
6.3
7.3
6.5
6.5
6.1
7.4
6.3
6.1
6.2
6.5
6.8
-18.7 1.9
-16.0 0.7
-19.1 1.6
-18.5 2.0
-16.3 1.3
-19.2 1.9
-17.4 1.8
-18.6 1.8
-17.1 1.1
-19.6 1.3
-17.7 1.3
-14.5
3.0
1.7
2.7
3.1
1.8
2.9
3.3
3.0
b
2.7
3.2
3.0
5.3
3.5
3.7
1.8
1.9
4.1
2.0
1.5
4.3
2.3
2.1
1.8
3.9
2.2
2.8
5.3
4.0
2.6
-10.5
-10.2
-12.8
-10.4
88
100
67
85
-10.2 >100
It appears that the compound 10 and one crystallo-
graphical independent form of AZT (molecule 1) have
close conformational parameters. In both cases an anti
conformation about the N-glycosidic bond and C2′-
endo-C3′-exo conformation of the furanose ring are
found in these molecules. Atoms C2′ and C3′ are
deviated from the atom planes C1′, C4′, O4′ for 0.307
and 0.181 Å in molecule 10 and for 0.352 and 0.170 Å
in the AZT molecule 1. The difference observed in the
conformation about the exocyclic C4′-C5′ bond can be
due to intermolecular interactions within the crystals.
Another group of conformationally similar molecules
includes the oximes 3E and 5Z (Table 3). The glycosidic
torsion angles and conformations about the exocyclic
C4′-C5′ bonds in these compounds are coincidental with
each other, and the furanose ring puckering belongs to
the C1′-exo population. In the oxime 3E the furanose
cycle C1′-exo-O4′-endo (1To) conformation is realized.
Atoms C1′ and O4′ are deviated to the opposite sides of
the plane of atoms C2′, C3′ and C4′ for 0.292 and 0.087
Å, respectively. Atoms of the 3′-oximino group (N3′O6′H)
are in the plane of atoms C2′, C3′, and C4′. The value
of the glycosidic torsion angle for 3E is -118.1°, and
this value is rather close to the angle of AZT molecule
1 (-125.4°). Always in the case of 3E, and with the aim
to compare the solid state and solution structure, it was
not possible to perform complete pseudorotational analy-
sis in order to determine conformation of the sugar ring
-13.0
-12.6
-10.7
-10.4
-16.0
-12.9
-10.8
-10.5
-12.6
-12.4
63
80
85
69
81
77
28
60
72
9c
-15.0
-15.0
10c
11d
-18.4 3.0
a
b
c
J 2′′,4′) 1.0 Hz. Signal is overlapped. J 2′,3′ ) 8.2, 7.9, and
8.1 Hz; J 2′′,3′ ) 2.6, 2.6, and 3.5 Hz; J 3′,4′ ) 3.4, 6.2, and 4.1 Hz for
compounds 8, 9 and 10, respectively J 2′′,4′ ) 1.5 Hz.
d
These features are conserved and were repeated in all
the other oximes (2E + 2Z, 4E + 4Z, 5E, 6E + 6Z, and
7E + 7Z) reported in this paper. On the basis of these
features, the assignment of E/Z configuration of those
other oximes has been carried out. Thus, for each E/Z
isomeric pair the chemical shift of the 2′′ proton was
always more downfield for the E-isomer, the chemical
shift of H4′ was usually more upfield for the E isomer
and the chemical shift of H6 was always more upfield
for the E isomer (Table 1); J 1′,2′ was bigger for the Z
isomer, J 1′,2′′ was bigger for the E isomer, and J 2′,2′ was
bigger for the E isomer. Also, the population of the γ+
rotamer around the exocyclic C4′-C5′ bond was always
bigger for the Z isomer (Table 2).
Regarding the nitro derivatives 8-10 (Scheme 2), the
assignment of a threo configuration for the minor
diastereomer 9 in the mixture of 8 + 9 was derived from
the “sum rule”8,19 based on the analysis of the exocyclic
1
3
using H NMR owing the absence of endocyclic J 2′,3′
,
3
3
J 2′′,3′, J 3′,4′ coupling constants. However, it has been
shown23 that the using of the equation
+
J 4′,5′ and J 4′,5′′ coupling constants using the equation Pγ
) (13.3 - ∑(J 4′,5′ + J 4′,5′′))/9.7.20 For the threo isomers
the population of the γ+ rotamer is usually less than
30%.8 In our case for the major 8 and minor 9 isomers
these calculations gave 77% and 29%, and this suggests
the erythro and threo configurations, respectively.
%N ) (7.9 - 3J 1′,2′) × 100/6.9
allows us to determine the percentage of the north
population (%N) in the two-state north-south equilib-

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Potential Chemotherapeutic Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 489
tions8,20,23 allow to determine the percentage of rotam-
ers:
x(γ+) ) (13.3 - 3J 4′,5′ + 3J 4′,5′′)/9.7
∑
3J 4′,5′ ) x(γ+)2.4 + x(γ-)10.6 + x(γi)2.6
x(γ+) + x(γ-) + x(γi) ) 1
3
3
The use of experimental J 4′,5′ and J 4′,5′′ coupling
constants (2.7 and 4.1 Hz, respectively) in these equa-
tions gave the relative percentages of the staggered
rotamers across C4′-C5′ as follows: γ+ ) 67%, γ-
)
3%, and γi ) 30%. The distributions obtained for the
E-oximes 5E and 7E were very similar (data are not
cited). However, the percentage of the γ+ rotamers for
the Z-oximes 5Z and 7Z were essentially bigger (5Z γ+
) 81%, γ- ) 11%, and γi ) 8%; 7Z γ+ ) 83%, γ- ) 10%,
and γi ) 7%). The preference of the γ+ rotamers is in
agreement with X-ray data for the oximes 3E and 5Z
where the γ+ conformation was found. Moreover, in
order to determine the conformation around the glyco-
sidic C1′-N1′ bond the approach proposed by Davies24
3
based on the analysis of experimental J H1′,C2 and
3J H1′,C6 coupling constants was applied. Using of the
experimental coupling constants obtained for 3E (3J H1′,C2
3
) 2.6 Hz and J H1′,C6 ) 4.1 Hz) in the following
equations
J ≈ (A2 + A6)cos2 λ + (C2 + C6)
∑
P ) 0.5 - {(∆J - ∆C)/2 B cos λ} +
∑
a
{∆A cos λ/2 B}
∑
where Pa is the population of anti conformer, ∑J )
3
3J H1′,C2
+
3J H1′,C6, ∆J ) J H1′,C6
-
3J H1′,C2 and the
coefficients A, B, and C are the experimental Karplus
parameters determined for a number of uridine deriva-
tives (A2 ) 5.0, A6 ) 6.2, C2 ) 0.1, C6 ) 0.1, B2 )
-2.1, B6 ) -2.4 Hz), allowed to determine the popula-
tion of anti conformer Pa ) 0.62, suggesting predomi-
nant anti conformation of 3E which is typical for
pyrimidine nucleosides [the determined value of Pa for
3
the natural thymidine (3J H1′,C2 ) 2.2 Hz, J H1′,C6 ) 3.8
Hz) is 0.65 (error in these calculations is (0.02)].
For the methylated oxime 5Z, a C1′-exo-C2′-endo
(2T1) conformation is observed with C1′ and C2′ atom
deviations of 0.183 and 0.302 Å, respectively, to opposite
sides of the C3′, C4′, and O4′ atoms plane. In spite of
the presence of a double bond between the C3′ and N3′
atoms, the N3′ atom is displaced by 0.312 Å from the
plane of atoms C3′, C4′ and O4′, and the 3′-methoxy-
imino group forms, with this plane, an angle of 26°. The
deviation of the atoms of the methoxyimino group from
the furanose cycle plane is probably due to the involve-
ment of the N3′ atom in intramolecular hydrogen bonds
with N3 atom of the adjacent molecule.
F igu r e 2. Three-dimensional structure of the crystal confor-
mation of 3E, 5Z (and 1010).
3
rium on the basis of J 1′,2′ coupling constant. This
3
calculation for 3E with experimental value of J 1′,2′
)
6.3 Hz (D2O) gave 23% for north and 77% for south
conformer, respectively. Predominance of south popula-
tion in solution was in accordance with X-ray data. The
similar results were obtained for the E-oximes 5E and
7E (data are not cited). The conformation around the
exocyclic C4′-C5′ bond (γ) can be described through
relative distribution (x) of three staggered rotamers
population γ+, γ-, and γi and the following equa-
The conformation of the sugar ring of the oximes 3E
and 5Z is exceedingly different from the conformations
found in natural thymidine and in AZT. Some flatten-
ing of the furanose cycle is observed for all compounds
studied, comparable to thymidine. This flattening is
especially apparent for the oxime 3E where the degree
of pucker is 25.7°. The same parameter for thymidine
is 37.8°.

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490 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ta ble 3. Conformational Parameters of Some Nucleoside Analogues Obtained from X-ray Studiesa
3E 5Z 10 AZT molecule 1 AZT, molecule 2
Fedorov et al.
Thd
conformation about
N1′-C1′ bond
ø (O4′C1′N1C2), deg
P (Phase angle of
pseudorotation), deg
anti
anti
anti
anti
anti
anti
-118.1
115.6
-118.9
147.7
-121.9
174.9
-125.4
173.3
-172.0
212.2
-139.4
187.5
Ψm (degree of pucker), deg 25.7
31.2
30.5
32.4
36.3
37.8
furanose ring conformation C1′-exo/O4′-endo C1′-exo/C2′-endo C2′-endo/C3′-exo C2′-endo/C3′-exo C2′-endo/C3′-exo C2′-endo/C3′-exo
conformation about
C4′-C5′ bond
gauche+
gauche+
gauche-
gauche+
trans
trans
γ (O5′C5′C4′C3), deg
43.1
48.8
-71.9
50.8
173.5
172.8
a
Conformational parameters for 10,10 thymidine21 (Thd), and AZT22 (molecules 1 and 2) were calculated from coordinates taken from
above mentioned references.
Ta ble 4. Anti-HIV-1 and -HIV-2 Activity and Cellular Toxicity of Nucleoside Analogues in Human MT-4 and CEM Cellsa
EC50, µg/mLb
HIV-1
CEM
0.40
26
1.0
g100
13
HIV-2
CEM
0.35
27
0.80
100
10
0.0009
CC50, mg/mLc
CEM
CEM/TK- d
9.9
>200
104
selectivity index HIV-1 and HIV-2
compd
3E
5E + 5Z 3.3
7E + 7Z 0.05
10
11
AZT
MT-4
0.025
MT-4
CEM/TK- MT-4
MT-4
CEM
0.03
4.6
0.05
2.2
20
>100
>20
1.6
182
7.1
93
91
>100
>200
>200
>200
>200
>200
35-88
34-64
25-28
8
120-182
48-79
104-130
g1.7
19-25
>100000
1.0
0.86
0.0005
>100
>100
>25
168
247
1.5 >100
1.2
77-102
2100-3000
0.0007 0.0007
a
Experiments were performed with HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 or CEM cells. The values reported are the average from
b
two independent experiments. Selectivity index was calculated as follows: SI ) average CC50/average EC50
.
50% effective concentration
or compound concentration required to protect MT-4 cells against cytopathogenicity of HIV or to protect CEM cells against HIV-induced
giant cell formation by 50%. c 50% cytotoxic concentration or compound concentration required to reduce MT-4 cell viability by 50%.
CEM/TK-: thymidine kinase deficient CEM cells.
d
An tivir a l Activity a n d Cellu la r Toxicity. Com-
pounds 3E, 5E + 5Z, 7E + 7Z, 10, and 11 were
evaluated for their inhibitory activity against HIV-1 and
HIV-2 in cell cultures (Table 4). Compound 3E and the
3′-acetyl derivative 7E + 7Z proved markedly inhibitory
to HIV-1 and HIV-2 replication in MT-4 and CEM cell
cultures. The antiviral potency of these test compounds
against HIV-1 and HIV-2 proved 10-20 fold higher
when evaluated in MT-4 cell cultures than in CEM cell
cultures. Also, the cytostatic activity of the test com-
pounds were 5-15-fold higher against MT-4 than
against CEM cells (Table 4). The methyl derivative 5E
+ 5Z proved less inhibitory to HIV-1 and HIV-2 by
approximately 2 orders of magnitude. The 3′-nitro (10)
and 3′-nitronate (11) derivatives showed an EC50 in the
range of 1 µg/mL in MT-4 cell cultures and 10-100 µg/
mL in CEM cell culture.
The compounds were also evaluated against a panel
of DNA and RNA viruses, including herpes simplex
virus type 1 (HSV-1), HSV-2, the thymidine kinase
deficient strain of HSV-1 (B2006), vaccinia virus (VV)
and vesicular stomatitis virus (VSV) in human E6SM
cells, Sindbis virus, Semliki forest virus, parainfluenza
virus, Coxsackie virus and reovirus-1 (Reo-1) in Vero
cells, Coxsackie virus and VSV in Hela cells, and
varicella-zoster virus (wild-type strains OKA and YS,
and TK- strains 07/1 and YS:R) and cytomegalovirus
(strains AD169 and Davis) in human embryonic HEL
cells. None of the compounds were markedly inhibitory
at 200 µg/mL against parainfluenza virus, reovirus-1,
Sindbis virus, Coxsackie virus B4, Semliki forest virus,
vesicular stomatitis virus, and vaccinia virus, except for
compound 7E + 7Z that had a MIC50 of 70 µg/mL
against VV, and compound 3E that had a MIC50 of 135
µg/mL against VSV. Compounds 3E and 7E + 7Z
showed marginal activity against VZV (OKA strain)
(MIC50: 11-20 µg/mL).
In contrast, compounds 3E and 7E + 7Z proved
markedly inhibitory to the replication of several HSV-1
strains (EC50: 0.4-1.3 µg/mL for 3E and 0.9-5 µg/mL
for 7E + 7Z) (Table 5). Also, these compounds proved
inhibitory to HSV-2 strains, albeit at a lower potency
than for HSV-1. Compounds 3E and 7E + 7Z proved
inactive against the thymidine kinase deficient (TK-)
HSV-1 strain B2006 (Table 5).
Striking differences were found with regard to the
cytostatic activities of the test compounds depending the
cell type against which they were evalutated. Whereas
the cytostatic effects of the test compounds were similar
for L1210, Molt4 (clone 8 and MT-4) cells (Tables 4 and
6), their inhibitory potential to CEM cell proliferation
was markedly lower, and no inhibitory activity was
found against murine mammary carcinoma FM3A cells
at 200 µg/mL (Table 6). Since compounds 3E and 7E
+ 7Z showed a marked inhibitory effect on the replica-
tion of TK+ HSV-1 strains (KOS, F, Mc Intyre), but not
the TK- HSV-1 strain B2006, they were also evaluated
for their inhibitory effect on FM3A cells transfected by
the HSV-1 TK gene (Table 6). Howewer, neither 3E nor
7E + 7Z or the other test compounds (5E + 5Z, 10, 11)
proved active against FM3A/TK-/HSV-1 TK+ cells.
Compounds 3E, 5E + 5Z, and 10 were also evaluated
for their in vitro anti-HBV activities in the HBV-
transfected 2.2.15 cell line. The derivative 3E demon-
strated a significant anti-HBV activity with a 50%
effective concentration (EC50) of 0.25 µg/mL (1 µM) for
inhibiting intracellular viral replicative intermediate
DNA as compared to control. In contrast, the com-
pounds 5E + 5Z and 10 exhibited no in vitro anti-HBV
activity up to a concentration of 10 µM. None of these
compounds exhibited in vitro cytotoxicity in Hep-G2
cells up to a concentration of 50 µg/mL (200 µM), thus
demonstrating the important anti-HBV selectivity (>200)
of the 3E derivative.

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Potential Chemotherapeutic Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 491
Ta ble 5. Cytotoxicity and Antiviral Activity of Nucleoside Analogues in E6SM and in HEL Cells
50% minimum inhibitory concentration (µg/mL)b
minimum
HSV-1
(KOS)
HSV-1
(Mc Intyre)
HSV-2
(G)
HSV-2
(Lyons)
HSV-1 TK-
(B2006)
cytotoxic
concentrationa
(µg/mL)
HSV-1 (F)
E6SM
HSV-2 (196)
E6SM
compd
3E
5E + 5Z
7E + 7Z
10
E6SM
0.4
>200
0.9
>200
35
HEL
E6SM
HEL
E6SM
HEL
E6SM
HEL E6SM HEL
>400
>200
400
>200
>200
g300
>400
>100
g400
1.4
50
0.5
>200
5
1.3
>200
2
>200
35
0.85
>50 >200
2.7
0.7
0.5
>50
2.8
20
ND
30
11
>200
70
>200
>200
>400
150
1.4
>200
4
>200
>200
>400
100
0.31 g200 >50
35
2
25
ND
5
>200 >50
>200 >5
>200 >50
>200 ND
10 50
3.8
5
1
>200
>200
90
>200
20
11
NDc
9
ND
0.003
ND
BVDU
ribavirin
DHPG
ACG
0.007 0.005
0.02
80
0.002
0.02
90
90
60
ND
0.001 ND
0.01 ND
50
ND
ND
150 ND
0.003 ND
0.006 ND
0.002 ND
0.009 ND
0.006
0.02
0.002 ND
0.004 ND
5
ND
100 ND
a
Minimum cytotoxic concentration that causes a microscopically detectable alteration of normal cell morphology after 2 days of incubation.
Concentration required to reduce virus-induced cytopathogenicity by 50%. c ND, not determined.
b
Ta ble 6. Inhibitory Effects of Nucleoside Analogues on the
Proliferation of Murine Leukemia (L1210), Murine Mammary
Carcinoma Cells (FM3A), and Human T-Lymphocyte (Molt4/C8)
Cells
methyl 2,3-dideoxy-3-nitro-5-O-toluoyl-R/â-D-erythro-
pentofuranoside25 which was consequently condensed
with silylated thymine to afford an R and â anomeric
mixture of only the erythro nucleosides.8 Deprotection
of all these products under acidic or basic conditions
gave 3′-nitro-3′-deoxythymidine with the natural erythro
configuration.8 The same conclusion that only erythro-
3′-nitro-3′-deoxythymidine 10 is stable was confirmed
by the recent synthesis of this compound by the reaction
of mixture of erythro- and threo-1-(2-deoxy-5-O-trityl-
3-deoxy-3-iodopentofuranosyl)-2-methoxy-5-methyl-4(1H)-
pyrimidinone with LiNO2 following by deprotection in
acidic conditions to give exclusively 10.9 On the other
hand, it has been previously reported that the oxidation
of sugar 3-oximes24 and nucleosides,8 bearing appropri-
ately protected hydroxyl functions at C2′, provides the
formation of mixtures of erythro and threo compounds.
EC50, µg/mLa
FM3A/TK-
compd
3E
5E + 5Z 112 ( 60
7E + 7Z 19 ( 6
10
11
L1210
FM3A FM3A/TK- HSV-1 TK+
Molt4/C8
1.1 ( 0.3 >200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
1.2 ( 0.4
>200
14 ( 3
70 ( 19
17 ( 5
>200
>200
>200
>200
58 ( 2
25 ( 5
a
50% cytotoxic concentration or compound concentration re-
quired to reduce cell viability by 50%.
Discu ssion
Ch em istr y. The synthetic precursor of the 3′-N-
hydroxyimino derivative 3E, namely the 5′-protected 3′-
ketothymidine 1 (Scheme 1) is an unstable compound
which easily undergoes â-elimination in the presence
of protic solvents. However, the nitrogen of the oxime
group of 3E is a much weaker electron acceptor than
that of the oxygen of the keto group of 1 which provides
strongly increased oxime stability. Thus, only traces
of thymine were observed after deprotection of the
oximes 2E + 2Z under acidic conditions. An aqueous
solution of the oxime 3E is stable at room temperature
for several months, and no traces of thymine formation
have been observed. The acetate 7E + 7Z appears to
be hydrolytically unstable, releasing parent 3E, and its
half-life in water solution did not exceed 24 h. This
process is accompanied by strong decomposition with
release of thymine (data are not cited).
It was surprising that the oxidation of the 5′-protected
oximes 2E + 2Z by CF3COOOH in acetonitrile in the
presence of Na2HPO4 gave a mixture of erythro and
threo nucleosides 8 and 9 in a ratio of 7:1. The exact
reasons for the formation of the 5′-tritylated threo-3′-
nitro-3′-deoxythymidine 9 during this oxidation are not
clear. Probably it is due to the steric hindrances from
the â-face of the oximes 2E + 2Z, where are disposed
the bulky nucleic base and MMTr group, and which
might bring about the energetically unfavored threo
configuration. However, deprotection of the diastereo-
meric mixture of 8 + 9 afforded only the expected
erythro-3′-nitro-3′-deoxythymidine (10), and this sug-
gests a total instability of threo-3′-nitro-3′-deoxythymi-
dine in protic solvents.
The 5′-monomethoxytritylated oxime 2E + 2Z exists
in organic solvents as a pair of E/Z diastereomers.
However, after deprotection with 80% aqueous acetic
acid the only product identified in aqueous solution was
the E isomer 3E (1H NMR data). Nevertheless, in
organic solvents such as DMSO-d6 and CD3OD, 3E
immediately generates the corresponding Z isomer, and
Str u ctu r e-Activity Rela tion sh ip . The 3′-N-hy-
droxyimino derivative 3E represents a novel inhibitor
of HIV and HSV replication in cell culture. It does not
discriminate between HIV-1 and HIV-2 (Table 4) and
is also active against both HSV-1 and HSV-2 (Table 5).
The compound proved virtually inactive against VZV
and CMV in HEL cells. It is noteworthy that com-
pounds 3E and 7E + 7Z are about as active against
HSV-1 and -2 in HEL cells as in E6SM cells, which thus
means that their inactivity against VZV in HEL cells
must be ascribed to the virus and not to the cells. The
inactivity of 3E and its closely related 7E + 7Z deriva-
tive against VZV is somewhat surprising in the light of
the close similarities of substrate properties between
HSV-1 thymidine kinase (TK) and VZV thymidine
kinase. Indeed, the observation that 3E and 7E + 7Z
are inactive against a thymidine kinase deficient HSV-1
1
a mixture of E and Z isomers can be observed by H
NMR (data are not cited).
The 3′-nitro derivative 10 has been previously syn-
thesized by a few independent synthetic routes. These
methods include (i) direct oxidation of 5′-toluoyl-3′-
amino-3′-deoxythymidine by CF3COOOH in acetonitrile
in the presence of Na2HPO4;8 (ii) reduction of 5′-
(monomethoxytrityl)-3′-nitro-3′-deoxy-2′,3′-didehydrothy-
midine by NaBH4 in ethanol;8 (iii) reduction of methyl
2,3-dideoxy-3-nitro-5-O-toluoyl-2,3-didehydro-R/â-D-pento-
furanoside by NaBH4 in ethanol to give exclusively

+
+
492 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Fedorov et al.
strain suggests that the compound 3E is dependent on
the phosphorylation by HSV-1 (and HSV-2) TK to
become active against the virus. Therefore, one may
conclude that the TKs of HSV-1 and VZV are endowed
with different affinities for these test compounds.
However, the inactivity of 3E and 7E + 7Z against VZV
(and CMV) can also be explained by differences in
cellular metabolism of the test compounds. Indeed, the
different anti-HIV properties depending the cell line
used in the anti-HIV assays (i.e. MT-4 or CEM), as well
as the difference in cytostatic activity of the compounds
against cell lines of different origin strongly suggest that
cellular metabolism may be an important determinant
in the eventual cytostatic and antiviral activity of these
test compounds. Likewise, the inactivity of 3E and 7E
+ 7Z against HSV-1 TK gene transfected FM3A cells
may be explained by such cellular factors, including
inactivity of the phosphorylated products against the
putative cellular target enzymes (i.e. dTMP synthase,
DNA polymerases) for cytostatic activity.
The additional clue that a flattened sugar configura-
tion could be preferable for sufficient recognition of
modified nucleosides and/or nucleoside 5′-triphosphates
by cellular and viral enzymes came from a comparison
of the activity of 3′-nitro (10) and the corresponding
nitronate (11) derivatives. Thus, 10 is inactive against
HIV-1 and HIV-2 in CEM cell cultures (EC50 g100 µg/
mL for HIV-1 and HIV-2), but the nitronate 11 shows
moderate activity (EC50 13 and 10 µg/mL for HIV-1 and
HIV-2, respectively) in the same cell line (Table 4). Both
the nucleoside analogues 10 and 11 show similar
cytotoxicity against CEM cells (168 and 247 µg/mL for
10 and 11, respectively) (Table 4), which suggests that
both of them are phosphorylated at least to the 5′-
monophosphate by cellular thymidine kinase. Then the
5′-monophosphate of 11 is probably consequently phos-
phorylated to the corresponding 5′-triphosphate to
inhibit the retroviral reverse transcriptase. As was
previously reported, the 5′-triphosphate of 3′-nitro-3′-
deoxythymidine 10 is a potent terminator of DNA chain
elongation, catalyzed by HIV-1 and HIV-2 RT,10 an
observation which suggests that a loss of activity of 10
by 1 order of magnitude (compared to 11) is likely due
to the poor phosphorylation of 3′-nitro-3′-deoxythymi-
dine 5′-monophosphate to the corresponding 5′-di- or
-triphosphate. A similar observation has been made for
the activity of 10 and 11 against herpes simplex virus
replication. Compound 10 is inactive against both
HSV-1 and HSV-2 in E6SM cells (EC50 >200 µg/mL),
whereas 11 shows moderate activity against several
HSV-1 strains (Table 5). Both 10 and 11 are devoid of
toxicity against the E6SM cell line. Compound 11 may
be still able to pass through all steps of activation by
cellular kinases, whereas compound 10 cannot. These
data demonstrate that recognition of two very similar
nucleoside analogues 10 and 11 by cellular and/or viral
enzymes of nucleic acid metabolism is preferable for 11,
having a more flattened sugar conformation, isosteric
to that of the very active oxime 3E, and thus supports
the design of novel molecules wherein such a conforma-
tion is realized.
replication in cell culture. In addition it is notworthy
that this compound is also active against HBV in HBV-
transfected 2.2.15 cell line. It would now seem impor-
tant to know how this compound exerts its biological
activities. In this regard, the mechanism of action of
3E is currently under investigation. Other work in
progress in our laboratories is directed toward the
application of the synthetic approach described above
to the preparation of various other 2′- and/or 3′-oximino
nucleoside analogues.
Exp er im en ta l Section
A. Syn th esis. Ma ter ia ls a n d Meth od s. The 1H NMR
and 13C spectra were recorded on a Bruker AC 250 spectrom-
eter at 25 °C in CDCl3 or D2O using TMS or MeCN as an
internal standard. The accepted abbreviations are as fol-
lows: s, singlet; dd, doublet of doublets; ddd, doublet of
doublets of doublets; m, multiplet; q, quartet; pt, pseudotriplet.
FAB mass spectra were recorded in the positive or negative
ion mode on a J EOL DX 300 mass spectrometer operating with
a J MA-DA 5000 mass data system using 3-nitrobenzyl alcohol
(NBA) as matrix. The UV spectra were recorded on a Uvicon-
931 spectrometer in water. TLC was performed on aluminium
silica gel F254 sheets (Merck, Art. 5554), visualization of
products being accomplished by UV absorbance followed by
charring with 10% ethanolic sulfuric acid with heating.
Column chromatography was carried out on silica gel 60
(Merck, Art. 15111), using methylene chloride and methanol
as eluents. Melting points were determined with a Reichert
melting point apparatus (Austria) and are uncorrected. Sepa-
ration of analytical samples of isomeric mixtures was per-
formed on precoated silica gel 60 F254 TLC plates (layer
thickness 0.5 mm, Merck, Art. 1.05744). Reverse phase
chromatography was performed on LiChroprep RP-18 (40-
63 µm, Merck, Art. 13900). Compound 1 was prepared as
previously reported.17 The X-ray analyses were carried out
on a CAD-4 diffractometer. The structures were solved by a
direct method and refined by the full-matrix least squares with
anisotropic approximation for nonhydrogen atoms. The hy-
drogen atom coordinates were determined from the difference
of Fourier syntheses and refined using the isotropic temper-
ature factors. Final values of R factors were 4.2% and 3.0%
for compounds 3E and 5Z, respectively. Crystals of all
compounds were obtained from water.
1-[5-O-(4-Mon om e t h oxyt r it yl)-2,3-d id e oxy-3-(N -h y-
d r oxyim in o)-â-D-glycer o-p en tofu r a n osyl]th ym in e (2E +
2Z). To a saturated solution of hydroxylamine hydrochloride
in pyridine (5 mL) was added compound 117 (1.45 g, 2.83
mmol). After 15 min, the reaction mixture was evaporated
and partitioned between water (50 mL) and dichloromethane
(50 mL). The organic layer was dried with anhydrous Na2-
SO4, evaporated, and re-evaporated with toluene. Column
chromatography on silica gel (stepwise gradient of MeOH in
CH2Cl2, 0 f 2.5%) gave 1.39 g of a mixture of 2E + 2Z as a
white foam (93%): MS m/e (FAB MS < 0, NBA) 527 (M - H)-.
1-[2,3-Did eoxy-3-(N-h yd r oxyim in o)-â-D-glycer o-p en to-
fu r a n osyl]th ym in e (3E). A solution of 2E + 2Z (193 mg,
0.37 mmol) in 80% aqueous acetic acid (5 mL) was stirred
overnight, evaporated to dryness, and re-evaporated with
toluene. The residue was partitioned between water (5 mL);
and methylene chloride (5 mL), the water phase was washed
with methylene chloride (5 mL), filtered through a wet paper
filter, and evaporated to dryness. The residue was puri-
fied by reverse phase chromatography (gradient of MeOH
in water, 0 f 5%) to give after freeze drying 64 mg of 3E
as a white foam (68%). Crystallization from water gave 43
mg of crystalline 3E: mp 117-119 °C; UV λmax 267 nm (ꢀ
9600); 13C NMR (20% aqueous CD3OD) δ 168.2 (C4), 161.6
(C3′), 153.4 (C2), 139.2 (C6), 113.8 (C5), 85.1 (C1′), 81.0 (C4′),
62.4 (C5′), 34.9 (C2′), 12.6 (Me-C5); MS m/e (FAB MS < 0,
NBA) 254 (M - H)-; (FAB MS > 0, NBA) 256 (M + H)+. Anal.
(C10H13N3O5‚H2O) C, H, N.
Con clu sion
We have shown that the hitherto unknown 3′-N-
hydroxyimino 3E analogue of thymidine is a novel
inhibitor of both HIV-1, HIV-2, HSV-1, and HSV-2
1-[5-O-(4-Mon om eth oxytr ityl)-2,3-d id eoxy-3-(N-m eth -
oxyim in o)-â-D-glycer o-pen tofu r an osyl]th ym in e (4E + 4Z).

+
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Potential Chemotherapeutic Agents
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 493
The reaction of a saturated solution of O-methylhydroxylamine
hydrochloride in pyridine (2 mL) with 1 (451 mg, 0.88 mmol),
followed by workup and purification on silica gel (stepwise
gradient of MeOH in CH2Cl2, 0 f 2%) as described for 2E +
2Z, gave 431 mg of 4E + 4Z as a white foam (91%): MS m/e
(FAB MS < 0, NBA) 540 (M - H)-.
1-[2,3-Did eoxy-3-(N-m eth oxyim in o)-â-D-glycer o-p en to-
fu r a n osyl]th ym in e (5E + 5Z). A solution of 4E + 4Z (222
mg, 0.41 mmol) in 80% aqueous acetic acid (5 mL) was stirred
overnight and worked up as described for 3E. Purification by
chromatography on silica gel (stepwise gradient of MeOH in
CH2Cl2, 0 f 7%) gave 79 mg of a mixture of 5E + 5Z as a
white foam (72%). Crystallization from water gave 45 mg of
crystalline 5Z: mp 121-123 °C. UV λmax 267 nm (ꢀ 9600);
13C NMR (20% aqueous CD3OD) (5E) 166.9 (C4), 159.5 (C3′),
152.0 (C2), 112.1 (C5), 84.6 (C1′), 79.8 (C4′), 62.3 (C5′), 61.1
(OMe), 33.1 (C2′), 11.9 (Me-C5); (5Z) 166.8 (C4), 159.8 (C3′),
152.1 (C2), 137.7 (C6), 112.7 (C5), 83.4 (C1′), 79.6 (C4′), 62.4
(C5′), 60.8 (OMe), 35.0 (C2′), 12.0 (Me-C5); MS m/e (FAB MS
< 0, NBA) 268 (M - H)-; (FAB MS > 0, NBA) 270 (M + H)+.
Anal. (C11H15N3O5) C, H, N.
1-[5-O-(4-Mon om et h oxyt r it yl)-2,3-d id eoxy-3-(N-a ce-
t oxyim in o)-â-D-glycer o-p en t ofu r a n osyl]t h ym in e (6E +
6Z). To a solution of a mixture of 2E + 2Z (460 mg, 0.87 mmol)
in dry pyridine (5 mL) with stirring at 0 °C was added acetyl
chloride (71 µL, 1 mmol). The reaction mixture was allowed
to warm up to room temperature, and after 6 h, a saturated
solution of NaHCO3 in water (2 mL) was added. The solution
was evaporated to dryness and worked up and purified on
silica gel (stepwise gradient of MeOH in CH2Cl2, 0 f 2%) as
described for 2E + 2Z to give 327 mg of a mixture of 6E + 6Z
as a white foam (66%): MS m/e (FAB MS < 0, NBA) 568 (M
- H)-.
1-[2,3-Did eoxy-3-(N-a cet oxyim in o)-â-D-glycer o-p en t o-
fu r a n osyl]th ym in e (7E + 7Z). A solution of a mixture of
6E + 6Z (340 mg, 0.60 mmol) in 80% aqueous acetic acid (5
mL) was stirred overnight and worked up as described for 3E.
The residue was purified on silica gel (stepwise gradient of
acetone in CH2Cl2, 0 f 50%) to give a mixture of 110 mg of
7E + 7Z as a white hygroscopic foam (62%). UV λmax 267 nm
(ꢀ 9600); 13C NMR (20% aqueous CD3OD) (7E) 172.0 [C(O)-
CH3], 168.9 (C3′), 166.9 (C4), 152.0 (C2), 138.3 (C6), 112.2 (C5),
84.9 (C1′), 80.0 (C4′), 62.1 (C5′), 34.5 (C2′), 19.0 [C(O)CH3],
11.9 (Me-C5); MS m/e (FAB MS < 0, NBA) 296 (M - H)-; (FAB
MS > 0, NBA) 298 (M + H)+. Anal. (C12H15N3O6‚H2O) C, H,
N.
1-[5-O-(4-Mon om eth oxytr ityl)-2,3-d id eoxy-3-n itr o-â-D-
er yth r o- a n d -th r eo- p en tofu r a n osyl]th ym in e (8 + 9). To
a stirred solution of a mixture of 2E + 2Z (393 mg, 0.75 mmol)
containing Na2HPO4 (5 g) and urea (10 mg) in acetonitrile (10
mL) at 0 °C was added dropwise a 4 M solution of pertrifluo-
roacetic acid in acetonitrile (60 mmol, 1 mL) during 15 min.
The mixture was allowed to warm up to room temperature,
and after 30 min the mixture was diluted with a saturated
aqueous solution of NaHCO3 (30 mL) and water (30 mL) and
extracted with methylene chloride (2 × 50 mL). The combined
organic layers were dried with Na2SO4, evaporated to dryness,
and purified on silica gel (stepwise gradient of MeOH in CH2-
Cl2, 0 f 2%) to give 255 mg of a mixture of the erythro and
threo isomers 8 + 9 in ratio 7:1 as a slightly yellowish foam
(62%): MS m/e (FAB MS < 0, NBA) 542 (M - H)-.
1-[2,3-Did e oxy-3-n it r o-â-D-er yt h r o-p e n t ofu r a n osyl]-
th ym in e (10). A solution of the diastereomeric mixture of 8
+ 9 (210 mg, 0.39 mmol) in aqueous 80% acetic acid (5 mL)
was stirred overnight and worked up as described for 3E.
Purification by reverse phase chromatography (stepwise gradi-
ent of MeOH in water, 0 f 7%) gave, after freeze drying, 85
mg of 10 as a white foam (80%): MS m/e (FAB MS < 0, NBA)
270 (M - H)-; (FAB MS > 0, NBA) 272 (M + H)+.
firmed by 1H NMR. MS m/e (FAB MS < 0, NBA) 270 (M -
Na+)-; (FAB MS > 0, NBA) 294 (M + H)+.
B. Exp er im en ts w ith cell cu ltu r es. Ma ter ia ls a n d
Meth od s. An tivir a l Activity Assa ys. The antiviral assays,
other than the anti-HIV-1 assays, were based on an inhibition
of virus-induced cytopathicity in either E6SM, HeLa, Vero, or
HEL cell cultures, following previously established proce-
dures.26-28 Briefly, confluent cell cultures in microtiter 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.
Cytosta tic Activity Assa ys. The cytostatic assays were
performed as previously described.29 Briefly, 100 µL aliquots
of the cell suspensions (5 × 105 murine leukemia L1210 or
murine mammary carcinoma FM3A or 7.5 × 105 human
T-lymphocyte Molt-4 or CEM cells/mL) were added to the wells
of a microtiter plate containing 100 µL of varying concentra-
tions of the test compounds. After a 2-day (L1210, FM3A) or
3-day (Molt-4 and CEM) incubation period at 37 °C in a
humidified CO2-controlled incubator, the number of viable cells
was determined using a Coulter Counter. Cytostatic activity
is expressed as the compound concentration that reduced the
number of viable cells by 50% (CC50). The cytotoxicity
measurements were based on microscopically visible alteration
of normal cell morphology (E6SM, HeLa, Vero) or inhibition
of normal cell growth (HEL), as previously described.28
In h ib it ion of H IV-In d u ced Gia n t Cell F or m a t ion .
CEM cell cultures were suspended at 250000-300000 cells/
mL of culture medium and infected with HIV-1 (IIIB) or HIV-2
(ROD) at 100 CCID50/mL. Then, 100 µL of the infected cell
suspension were transferred to 200 µL microtiter plate wells
containing 100 µL of serial dilutions of the test compound
solutions. After 4 days of incubation at 37 °C, cell cultures
were examined for syncytium formation as previously de-
scribed.30
An ti-Hep a titis B Vir u s Assa ys. The 2.2.15 HBV trans-
fected human hepatoma cells derived from the Hep-G2 cell line
were cultured as described by Korba and Guerin31 with minor
modifications. Cells cultured in Dubelcco’s modified eagle
medium supplemented with 4% fetal bovine serum and 0.5 mM
glutamine were treated with drugs for 9 days, and culture
medium was changed every 3 days. Hep-G2 cells and un-
treated 2.2.15 cells were used as negative and positive controls.
At harvest, the medium was removed and cells were lysed.
Total intracellular DNA was recovered and subjected to
southern blot analysis using a 32P-labeled HBV specific probe
(pTHBV plamid which contains the full length HBV genome)
kindly provided by Dr. Raymond F. Schinazi (Emory Univer-
sity, Atlanta, GA). Inhibition of the viral replicative interme-
diate DNA in drug-treated cells versus control was determined.
Evaluation of compound cytotoxicity was performed in Hep-
G2 cells by measuring the uptake of neutral red dye in a 96-
well cell culture plate. Cells were cultured and treated under
the same conditions as those used for evaluating the antiviral
activity.
Ack n ow led gm en t. These investigations were sup-
ported by grants from the CNRS, “Agence Nationale de
Recherches sur le SIDA” and the “Ministe`re de la
Recherche et de la Technologie, De´le´gation aux Affaires
Internationales” (France), as well as by the Russian
Program “National Priorities in Medicine and Public
Health: AIDS”, Grant SP O54, and by Russian Fond
for Fundamental Researches, Grant 96-04-4983. They
were also supported by the Biomedical Research Pro-
gramme of the European Community and by grants
from the Belgian Fonds voor Geneeskundig Weten-
schappelijk Onderzoek (project number 3.0180.95), the
Belgian Nationaal Fonds voor Wetenschappelijk Onder-
zoek (project number 3.3010.91), the Belgian Geconcer-
1-[2,3-Dideoxy-3-n itr on ate-â-D-glycer o-pen tofu r an osyl]-
th ym in e Sod iu m Sa lt (11). A solution of Na2CO3 (4.1 mg,
0.039 mmol) in water (0.5 mL) was added to 10 (12.5 mg, 0.045
mmol). After 1 h of stirring, and following freeze drying, 15.7
mg of a white foam was obtained which contained 90% of
nitronate 11 and excess of Na2CO3. Total conversion of 10 to
nitronate 11, which was not additionally purified, was con-

+
+
494 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
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