Preparation and anti-HIV activity of N-3-substituted thymidine nucleoside analogs

Article abstract of DOI:10.1021/jm9600095

A series of 22 derivatives of AZT substituted at the N-3 position of the thymine base were prepared and evaluated for anti-HIV activity in cell culture (Lai strain of HIV-1 in CEM-c113 cells). The AZT analogs bearing a N- 3 amino group (7), a hydroxyalkyl

Full text of DOI:10.1021/jm9600095

1550
J . Med. Chem. 1997, 40, 1550-1558
P r ep a r a tion a n d An ti-HIV Activity of N-3-Su bstitu ted Th ym id in e Nu cleosid e
An a logs
David R. Adams,† Caroline Perez,† Michel Maillard,† J ean-Claude Florent,‡ Michel Evers,§ Yvette He´nin,§
Simon Litvak,| Laura Litvak,| Claude Monneret,*,‡ and David S. Grierson*,†
Institut de Chimie des Substances Naturelles CNRS, Ave de la Terrasse, 91198, Gif-sur-Yvette, France,
Service de Chimie, CNRS, URA 1387, Institut Curie, Section de Biologie, 26 rue d’Ulm, Paris Cedex 05, France,
Institut de Biochimie et Ge´ne´tique Cellulaire, 1, rue Camille Saint-Saens, 33077 Bordeaux Cedex, France, and
Rhoˆne-Poulenc Rorer S.A., CRVA, 13 Quai J ules Guesde, BP 14, 94403 Vitry-sur-Seine Cedex, France
Received J anuary 2, 1996X
A series of 22 derivatives of AZT substituted at the N-3 position of the thymine base were
prepared and evaluated for anti-HIV activity in cell culture (Lai strain of HIV-1 in CEM-c113
cells). The AZT analogs bearing a N-3 amino group (7), a hydroxyalkyl chain (12f), and a
phosphonomethyl (12k ) substituent displayed activities in the 0.045-0.082 µM range. The
analogs 12d , 12e, 12q, 15, and 19 were active at <0.5 µM concentration. Compound 18 in
which two molecules of AZT are connected at N-3 via a two-carbon link and “dimer” 11 also
displayed significant activity. To obtain information concerning the mechanism of RT inhibition
by these AZT analogs, compounds 7, 12d , 12e, and 12q were incubated with recombinant HIV-1
RT in the presence of poly(A)-oligo[dT(12-18)] and poly(C)-oligo[dG(12-18)] template-primers. In
contrast to AZT-TP (control), none of these nucleosides displayed any significant inhibition of
RT in the recombinant enzyme assay, indicating that phosphorylation is a necessary prerequisite
for activity.
The discovery that the 2′,3′-dideoxynucleoside analogs
3′-azido-3′-deoxythymidine (AZT, 1),1 ddI (2),2 and ddC
(3)3 are potent inhibitors of HIV reverse transcriptase
(RT) marked a decisive advance in the search for anti-
AIDS drugs. However, the long-term administration of
these compounds to AIDS patients is limited by a num-
ber of severe side effects4 and by the appearance of drug
resistant HIV strains.5 In the search for new, longer
acting, more selective, and less toxic anti-HIV agents,
a wide range of 2′,3′-dideoxynucleoside analogs have
been prepared.6 Interested by structure-activity rela-
tion studies in this area, we initiated a program several
years back to examine the influence of substitution at
the N-3 position of the thymine base of AZT, and other
2′,3′-dideoxynucleoside analogs, on HIV replication.7-9
Assuming that such molecules inhibit RT in the same
manner as AZT itself, one might, a priori, anticipate
that N-3 substitution would alter base-base interac-
tions at several levels and, in particular, in their capaci-
ty to act as chain terminators.10 In initial experiments
it was found that, in contrast to the N-3 amino deriva-
tives of ddT (4), 3′-FddT (5), and D4T (6), which display
little or no activity, N-3 amino AZT 7 (RP67042)7,11
inhibited the HIV-induced cytopathic effect in CEM-
c113 cells 7 days after infection with an EC50 of 45 nM
and a selectivity index (SI ) CC50/EC50) of up to 10 000
as measured by the MIT method. Furthermore, al-
though 7 displayed reduced antiviral activity in vitro
(anti-HIV assay) and in vivo (anti-FR-MuLV assay)
relative to ACT (EC50 ) 0.003 µM), it was determined
to be potentially less toxic in vivo and to have a longer
plasmatic half-life in mice.
Building upon these observations, we describe in the
present paper the synthesis and evaluation for anti-HIV
activity in cell culture (Lai strain of HIV-1 in CEM-c113
cells) of a series of 22 new AZT analogs (compounds 11,
12a -q, 15, 17, 18, and 19) bearing structurally and
electronically diverse substituents at the N-3 position
of the thymine ring.
Ch em istr y
The AZT employed in this work was prepared by the
reaction of the 5′-O-(thexyldimethylsilyl)-protected an-
hydrothymidine derivative 8 with NaN3 in hot DMF,
followed by O-deprotection of intermediate 9 (R )
thexyldimethylsilyl) (Scheme 1).12 Although an excess
of azide ion was generally used to obtain 9, on several
occasions formation of minor amounts of the dimer 10
was also observed. The structure of this minor compo-
nent was deduced from its NMR and mass spectra. The
formation of 10 can be seen to arise from competitive
reaction of 8 with the N-3 anion of AZT, generated as
an intermediate during N3--promoted anhydronucleo-
side ring opening. This supposition was confirmed in
a separate experiment in which the N-3 anion of 9 (R )
thexyldimethylsilyl) was generated and reacted with 8
in the absence of added sodium azide. Although exten-
† Institut de Chimie des Substance Naturelles CNRS.
‡ Service de Chimie, CNRS.
§ Rhoˆne-Poulenc Rorer S.A.
| Institut de Biochimie et Ge´ne´tique Cellulaire.
X Abstract published in Advance ACS Abstracts, April 1, 1997.
S0022-2623(96)00009-X CCC: $14.00 © 1997 American Chemical Society

N-3-Substituted Thymidine Nucleoside Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1551
Sch em e 1
surprisingly, to formation of substantial amounts of a
product containing two molecules of alkylating agent.
With the exception of epoxide 12l, which on contact
with atmospheric moisture was observed to undergo
slow nucleophilic ring opening to form diol 14 (Scheme
2), the entire series of AZT derivatives 12 proved stable
under ambient conditions. For compound 12l, it is likely
that epoxide opening is assisted by participation of the
thymine ring carbonyl group at C-2 (or C-4) as il-
lustrated in the scheme. A similar mechanism involving
an O-alkylated pyrimidinium ion intermediate is pro-
posed to account for the formation of N-3 hydroxyethyl
derivative 12d in the reaction of O-5′ TBS AZT with
excess 1,2-dibromoethane and potassium carbonate in
hot methanol. These conditions were employed in an
attempt to improve the yield of bromoethyl AZT deriva-
tive 12g beyond that obtained by method A. In this
instance there was a simultaneous exchange of the
terminal bromo function by a hydroxyl group and
deprotection of the O-5′ silyl ether affording 12d directly
in 95% yield.
The N-3 bromoethyl-substituted analog 12g and its
O-5′ TBS derivative 16 were also further elaborated to
compounds 15, 17, and 18 (Table 2). Use of amberlite
IR 400 (N3-) resin provided azide 15 (91%), and reaction
of 16 with potassium phthalimide followed by HF
afforded 17 in high yield. Reaction of 16 with 9 (R )
TBS) gave the bridged dimer 18.
Finally compound 19, containing an isoxazoline sys-
tem attached to AZT at N-3 (Scheme 2), was prepared
in two steps (55% overall yield) involving a 1,3-dipolar
cycloaddition reaction between O-TBS 12c and the
nitrile oxide generated from the O-TBS derivative of
2-nitroethanol, and subsequent double deprotection.
sive decomposition of the anhydronucleoside occurred
under these conditions, significant quantities of dimer
11 were isolated after O-deprotection.
As was previously shown, N-3-substituted derivatives
of AZT can be prepared by reaction of anhydronucleoside
8 with an electrophile prior to addition of azide ion and
ring opening (Scheme 1).13 This approach works well
for the formation of compounds 7 and 12a . However,
for electrophiles larger than a methyl group, there is
competing N- and O-alkylation of 8, the latter reaction
leading to products wherein azide is introduced at C-1′
of the sugar component. For this reason, compounds
12a -q, 15, 17, 18, 19, and dimer 11 were prepared by
N-3 alkylation of AZT as a discrete step (see Tables 1
and 2 and Schemes 1 and 2).
Biologica l Resu lts a n d Discu ssion
For almost all cases, the preparation of the AZT
derivatives 12 was conveniently achieved by deproto-
nation of AZT with NaH in DMF at 20 °C for 1 h
(method A of Table 1) followed by addition of the
appropriate electrophile. Use of excess NaH/alkylating
agent led in some cases to the formation of small
amounts of N-3,O-5′-dialkylated AZT byproducts. For
the preparation of 12l, glycidyl triflate proved far
superior as an alkylating agent than the corresponding
tosylate. In certain instances the use of method A
failed, notably for the reactions with (diethylphosphono)-
methyl triflate and bromoacetaldehyde diethyl acetal
wherein AZT was recovered largely unreacted. This
problem was resolved using potassium carbonate in
DMF to deprotonate either AZT (method B) or its 5′-O-
(tert-butyldimethylsilyl)-protected derivative 9 (method
C). Analogs 12p and 12q were both prepared using
1-(2-bromoethyl)-4-methoxy-5-methylpyrimidin-2-one as
the electrophile. In these cases the method C route
proved advantageous, as it was more convenient to
separate chromatographically the common silylated
intermediate 13p from the slight excess of alkylating
agent prior to deprotection. The deprotection of 13p to
give 12p was achieved using either tetrabutylammo-
nium fluoride in THF or aqueous hydrochloric acid. The
latter conditions served to simultaneously deprotect the
O-5′ silyl ether and liberate the thymine ring carbonyl
leading to 12q. The use of 1-(2-bromoethyl)-5-meth-
ylpyrimidine-2,4-dione instead of the 4-methoxy analog
as an electrophile for direct preparation of 12q led, not
Following the same protocols used to evaluate the anti
HIV activity of the lead compound 7 (RP67042),7,11 the
N-3-substituted AZT analogs 12a -q synthesized in this
study were tested and compared to AZT for cytotoxicity
and the ability to inhibit HIV replication in CEM-c113
cells (Lai strain14). The HIV-1-induced cytopathic effect
(CPE) was monitored by the MTT viability assay to
determine EC50MTT and CC50 as previously described.15
HIV replication was also followed by measurement of
the reverse transcriptase activity in the culture super-
natant (EC50RT). The results obtained are shown in
Table 3.
Compound 12a , substituted at N-3 by a nonpolar
methyl group, exhibited an EC50 of 940 nM. Increasing
the length of the alkyl chain to n-pentyl (12b) offered
no improvement in anti-HIV activity. Indeed, in both
cases, the presence of an alkyl chain led to a 20-fold
decrease in activity compared to RP67042, 7. The
introduction of an allyl double bond (12c) or a terminal
bromine atom (12g) into the chain also leads to a
significant drop in activity. Contrasting with this
observation is the good level of activity observed when
an azide function is present in the ethyl chain as in
compound 15. The absence of activity for compound 12i
indicated that a benzyl group is not tolerated at the N-3
position. This may be a consequence of both steric and
electronic effects, as compounds 12o and 17, which are
also substituted by bulky and relatively nonpolar aro-
matic substituents, are inactive.

1552 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Adams et al.
Ta ble 1
Compounds 12a , 12c, and 12i have recently been
examined for their anti-HIV activity in MT4 cells by
Kitade et al.8 In their study, the N-3 allyl-substituted
analog 12c was found to display significant activity, but
in keeping with our findings 12i was also determined
to be inactive.
exception is the diethyl phosphonate analog 12k which
displays an EC50 of 0.071 µM.
Looking further to determine the extent to which
large substituents are tolerated at the N-3 position of
AZT, it was found that analog 12q, and especially
compound 18 where two AZT components are linked via
a two-carbon tether, are potent inhibitors, with EC50’s
of 0.48 and 0.7 µM, respectively. Impressive also was
the finding that the deprotected dimer 11, where a 3′-
deoxythymidine residue is attached directly to the N-3
position of AZT, displays significant capacity to inhibit
HIV-1 replication. However, for 12q activity was totally
lost on conversion of the C-4 amide carbonyl to the
corresponding O-methyl imidate, as in 12p .
Interestingly, the presence of a terminal hydroxy
group on the alkyl chain as in compounds 12d -f
restored activity to a level comparable to that of 7. In
fact, for this set of compounds, activity was observed to
increase with increasing chain length. The influence
of a terminal hydroxyl substituent on anti-HIV activity
was most striking for compound 19 (EC50 ) 0.5 µM),
which like 12o (EC50 ) 143 µM) contains a heterocyclic
component in the side chain. However, simply incor-
porating an oxygen atom into the N-3 substituent does
not appear to be responsible for this effect, as activity
dropped to the micromolar range for the (methoxy-
ethoxy)methyl ether 12h as well as for ketals 12m and
12n , epoxide 12l, and the methyl ester 12j. A noted
From the results obtained, it is clear that a varied
range of substituents can be introduced at the N-3
position of the pyrimidine ring in AZT. Indeed, 11 of
the 22 N-3 functionalized AZT analogs studied inhibited
HIV replication in vitro with 50% effective concentra-
tions in the micromolar range or lower. Of these,

N-3-Substituted Thymidine Nucleoside Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1553
Ta ble 2
Sch em e 2
manner to AZT? For AZT it has been clearly shown that
the drug must initially be phosphorylated by cellular
kinases and that it is AZT triphosphate which inhibits
RT through competitive binding to the DNA synthesis
site and by chain termination upon incorporation into
the growing DNA chain. Secondly, if these N-3-substi-
tuted analogues do bind to the DNA synthesis site, does
the added functionality on the base component spill into
empty space in the cleft normally occupied by the tem-
plate primer (or growing DNA chain), or does it interact
in some way with the neighboring “allosteric” binding
site?16 Thirdly, can the presence of the N-3 substitution
alter the manner in which these AZT analogues interact
with RT to the point where, like the HEPT and TSAO
series of compounds,17,18 they associate preferentially
with RT at the allosteric binding site?19
In view of both the bulky nature of the functionality
present at N-3 in certain of our ACT analogs, and the
substrate specificity generally displayed by cellular
kinases, it was not immediately obvious that our
compounds inhibit RT through an AZT-type mechanism.
Concerning this problem, Kitade et al.8 found that the
triphosphate derivatives of N-3 allyl, and several other
N-3 alkyl derivatives of AZT, are indeed moderate
inhibitors of RT in in vitro assays. However, as the
molecules studied by these authors correspond to only
weakly active members of our series, this point required
further evaluation. Thus, compounds 7, 12d , 12e, and
12q were incubated with recombinant HIV-1 RT20 in
the presence of poly(A)-oligo[dT(12-18)] and poly(C)-oligo-
[dG(12-18)] template-primers, and the activities were
monitored at varying concentrations (up to 100 µM). In
compounds 12d , 12e, 12q, 15, and 19 were active at
less than 0.5 µM concentration, and compounds 12f and
12k were essentially equipotent to the lead compound
7 (RP67042) in CEM-c113 cells (i.e. EC50’s ) 50 nM).
Having established that biological activity can be
maintained upon substitution of the N-3 position of AZT,
a number of fundamental questions arise. Firstly, do
these compounds interact with RT at the nucleotide
binding site, and if so, do they inhibit RT in a similar

1554 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Adams et al.
Ta ble 3. In Vitro Anti-HIV Activity of N-3-Substituted AZT
an internal standard. Chemical shift data are reported in
parts per million (δ in ppm) where s, d, dd, t, q, and m
designate singlet, doublet, doublet of doublets, triplet, quartet,
and multiplet, respectively. 13C NMR spectra where recorded
in CDCl3 on the same instruments. Flash column chroma-
tography was performed using Merck aluminoxid 90 (activity
II-III) or silica gel 60 (Art. 9385). In all cases the solvent
system used for the chromatographic separations was chosen
such that on TLC an Rf of 0.25-0.30 was observed for the
compound to be isolated. All microanalytical results for C, H,
and N are within (0.4% of the theoretical value.
P r ep a r a tion of Dim er 11. To a solution of 5′-O-thexy-
ldimethylsilyl AZT 9 (40 mg, 0.098 mmol) in DMF (1 mL),
stirred at room temperature (23 °C) under argon, was added
sodium hydride (2.90 mg, 0.117 mmol) followed, after 30-60
min, by 5′-O-(thexyldimethylsilyl)-protected anhydrothymidine
9 (53.7 mg, 0.147 mmol). The resulting mixture was heated
at 120 °C for 20 h. In instances where substantial amounts
of unreacted 9 remained (TLC), a further quantity of NaH (0.6
equiv) was added, and heating was continued for an additional
12 h. The solvent was then evaporated in vacuo, and a
preliminary separation of the O-5′-protected dimer 10 from
other components in the product mixture was achieved by
preparative layer chromatography (silica gel; EtOAc/heptane).
Compound 10 was then obtained pure (8 mg; 11%) after flash
column chromatography (silica gel; 45% ethyl acetate/55%
heptane). Subsequent treatment of this product with excess
TBAF and normal extractive workup provided dimer 11: 1H
NMR (1H-1H correlation; DMSO-d6) δ 11.25 (s, H-3), 7.80 (s,
2H, H-6, H-6′′), 6.57 (t, J ) 7.5 Hz, H-1′), 6.15 (t, J ) 5 Hz,
H-1′′′), 5.58 (m, H-3′), 5.25 (t, J ) 5 Hz, OH′′′), 4.98 (t, OH′),
4.39 (q, J ) 6 Hz, H-3′′′), 4.12 (m, H-4′), 3.84 (m, H-4′′′), 3.63
and 3.54 (2m, 4H, H-5′, H-5′′′), 2.50-2.18 (m, 4H, H-2′, H-2′′′),
1.84 (s, CH3), 1.79 (s, CH3); 13C NMR (DMSO-d6) δ 163.70 and
162.81 (C-2, 2′′), 150.46, 150.25 (C-4, 4′′), 136.71, 134.86 (C-6,
6′′), 109.62, 108.78 (C-5, 5′′), 84.76, 84.42, 84.20, 81.68 (C-1′,
1′′′, C-4′, 4′′′) 61.97, 60.5 (C-5′, 5′′′), 59.65 (C-3′′′), 50.71 (C-3′),
36.55, 34.81 (C-2′, 2′′′), 12.90, 12.14 (CH3).
Meth od A: 3-Am in o-3′-a zid o-3′-d eoxyth ym id in e (7). To
a solution of AZT (60 mg, 0.225 mmol) in DMF (3 mL), stirred
at room temperature (23 °C) under argon, was added sodium
hydride (6.47 mg, 0.270 mmol) followed, after 30-60 min, by
O-(2,4-dinitrophenyl)hydroxylamine (52.5 mg, 0.26 mmol).22
After an additional 18 h the solvent was removed in vacuo,
and the residue was flash column chromatographed (silica gel;
25% ethyl acetate/heptane) to afford compound 7 as a colorless
solid (54 mg; 85%): mp 75 °C (EtOH/Et2O); all spectral data
coincide with the results reported in ref 16.
3-Meth yl-3′-a zid o-3′-d eoxyth ym id in e (12a ). AZT (100
mg, 0.374 mmol), NaH (11 mg, 0.45 mmol), and CH3I (0.25
mL, 0.40 mmol). Purification by silica gel flash chromatog-
raphy (80% EtOAc/heptane). 12a : colorless oil (90 mg; 85%);
IR (CHCl3, cm-1) 3450, 2115, 1686, 1672, 1630; 1H NMR
(CDCl3) δ 7.37 (s, H-6), 6.05 (t, J ) 6 Hz, H-1′), 4.37-4.43 (m,
1H, H-3′), 3.77-4.02 (m, 3H, CH2-5′, H-4′), 3.34 (s, NCH3), 2.74
(br m, OH), 2.57 (ddd, J ) 14, 7.5, 6 Hz, 1H, H-2′), 2.40 (ddd,
1H, J ) 14, 6, 5 Hz, H-2′), 1.94 (s, CH3); 13C NMR (CDCl3) δ
163.49 (C-2), 150.64 (C-4), 134.27 (C-6), 109.53 (C-5), 86.12
(C-1′), 84.36 (C-4′), 61.39 (C-5′), 59.68 (C-3′), 37.38 (C-2′), 27.51
(NCH3), 12.87 (CH3); CI-MS m/z 282 (M + 1)+. Anal.
(C11H15N5O4) C, H, N.
Analogsa
prod
EC50MTT (µM)b
CC50 (µM)c
EC50RT (µM)d
SIe
7
0.045
0.940
0.890
32.0
0.300
0.150
0.082
2.670
5.600
NAf
1.470
0.071
6.80
1.30
5.00
450
355
>60
350
161
154
64
53
281
28
283
240
>62
>131
130
>288
29
0.5
10000
380
>67
11
537
1027
780
20
50
-
193
3380
9
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
11
NT
NT
65
0.09
0.12
1.2
NTf
140
NT
14
4.7
NT
13
100
26
2
-
60
1.5
143.0
NA
0.480
2.2
143
>29
NT
0.1
>29
66
30
15
17
18
19
0.260
110.0
0.700
0.500
0.003
60
227
36
26
>5
1.5
114.0
1.4
1.3
0.003
230
2
51
52
1667
AZT
a
All tests were carried out in triplicate, and the results
represent mean values for at least two experiments in three wells.
b
EC50MTT (50% antiviral effective dose): concentration (µM) that
reduces by 50% the HIV induced cytopathic effect. c CC50 (50%
cytotoxic dose): concentration (µM) required to reduce by 50% the
d
viability of noninfected treated cells. EC50RT: concentration (µM)
that reduces by 50% the HIV produced in the supernatant (see
ref 15). c SI (selectivity index): ratio of CC50 to EC50
active. NT: not tested.
.
f NA: not
contrast to AZT triphosphate, which was used as a
control, none of these nucleosides displayed any signifi-
cant inhibition of RT in the recombinant enzyme assays.
This result contrasts markedly with the activities
observed in cell culture and in an indirect fashion
strongly supports the hypothesis that N-3-substituted
analogs of AZT require transformation to the corre-
sponding triphosphate forms before becoming effective
inhibitors of RT. It is very probable therefore that, like
AZT, these compounds inhibit RT through competitive
binding in the catalytic site for DNA synthesis.
Recent molecular modeling studies carried out in our
laboratory in which thymidine triphosphate is fitted into
the binding site that it interacts correctly with the three
catalytic aspartates and two Mg2+ ions show that, in
fact, the N-3 position of thymidine points into the
enzyme cleft. Thus, at least in the absence of the
primer-template, the enzyme experiences little dif-
ficulty in accommodating functionality present at this
position. Further modeling experiments provide a
picture of the spatial relationship between this N-3
functionality and non-nucleoside inhibitors, such as
TIBO and nevirapine, which are bound in the adjacent
allosteric pocket.16 Together with recent X-ray crystal-
lographic results, this information serves as a valuable
guide in work underway to construct “mixed” site
inhibitors.21
3-Allyl-3′-a zid o-3′-d eoxyth ym id in e (12c). AZT (299 mg,
1.12 mmol), NaH (27 mg, 1.13 mmol), and allyl bromide (0.14
mL, 1.65 mmol). Purification by silica gel flash chromatog-
raphy (50% EtOAc/heptane). 12c: colorless oil (295 mg; 86%);
IR (neat, cm-1) 3453, 2106, 1705, 1665, 1635; 1H NMR (CDCl3)
δ 7.38 (s, H-6), 6.07 (t, J ) 6 Hz, H-1′), 5.95-5.79 (m, CHd),
5.29-5.16 (m, dCH2), 4.55 (d, J ) 5 Hz, NCH2), 4.45-4.38
(m, H-3′), 4.04-3.79 (m, 3H, CH2-5′, H-4′), 2.61-2.50 (m, 2H,
H-2′, OH), 2.45-2.35 (m, 1H, H-2′), 1.94 (s, CH3), 13C NMR
(CDCl3) δ 163.43 (CO), 150.81 (CO), 134.87 (C-6), 131.66
(CHd), 118.26 (dCH2), 110.62 (C-5), 87.53 (C-1′), 84.60 (C-
4′), 62.12 (C-5′), 60.04 (C-3′), 43.68 (NCH2), 37.48 (C-2′), 13.34
(CH3); EI-MS m/z 307 M+. Anal. (C13H17N5O4) C, H, N.
3-(2-Hyd r oxyeth yl)-3′-a zid o-3′-d eoxyth ym id in e (12d ).
AZT (260 mg, 0.973 mmol), NaH (28 mg, 1.16 mmol), and
Exp er im en ta l Section
Melting points were determined using a Reichert Thermovar
apparatus and are uncorrected. Mass spectra were obtained
on an MS-50 AEI (EI, 70 eV) or an MS-9 AEI (CI, isobutane)
spectrometer. 1H nuclear magnetic resonance (NMR) spectra
were recorded in CDCl3 (except where noted) on a Bru¨ker
spectrometer (200 or 250 MHz), using tetramethylsilane as

N-3-Substituted Thymidine Nucleoside Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1555
2-bromoethanol (0.128 mL, 1.8 mmol). Purification by silica
gel flash chromatography (EtOAc/heptane/MeOH, 7/3/1)).
12d : colorless oil (290 mg; 88%); IR (neat, cm-1) 3443, 2105,
1705, 1670, 1636; 1H NMR (CDCl3) δ 7.50 (s, H-6), 6.11 (t, J )
6 Hz, H-1′), 4.38 (m, H-3′), 4.20 (t, J ) 5 Hz, NCH2), 4.02-
3.75 (m, 5H, CH2-5′, H-4′, CH2OH), 3.08 (br s, OH), 2.87 (br s,
OH), 2.52-2.38 (m, 2H, CH-2′), 1.93 (s, CH3); 13C NMR (CDCl3)
δ 164.26 (CO), 151.40 (CO), 134.95 (C-6), 110.31 (C-5), 86.56
(C-1′), 84.62 (C-4′), 61.69 and 61.06 (C-5′, CH2OH), 59.85 (C-
3′), 43.73 (NCH2), 37.63 (CH2-2′), 13.16 (CH3); CI-MS m/z 312
(M + 1)+. Anal. (C12H17N5O5) C, H, N.
3-(3-Hyd r oxyp r op yl)-3′-a zid o-3′-d eoxyth ym id in e (12e).
AZT (1.60 g, 5.99 mmol), NaH (173 mg, 7.21 mmol), and
3-bromopropanol (0.82 mL, 9.03 mmol). Purification by silica
gel flash chromatography (2% MeOH/EtOAc). 12e: colorless
oil (1.22 g; 63%); IR (neat, cm-1) 3439, 2107, 1702, 1660, 1635;
1H NMR (CDCl3) δ 7.69 (s, H-6), 6.18 (t, J ) 6 Hz, H-1′), 4.39
(app q, J ) 6 Hz, H-3′), 4.08 (t, J ) 6 Hz, CH2N), 3.98-3.71
(m, 5H, CH2-5′, H-4′, 2 × OH), 3.56-3.49 (m, 2H, CH2OH),
2.43 (app t, 2H, J ) 6 Hz, CH2-2′), 1.92 (s, CH3), 1.85 (p, J )
6 Hz, CH2); 13C NMR (CDCl3) δ 163.85 (CO), 151.12 (CO),
134.91 (C-6), 110.14 (C-5), 86.36 (C-1′), 84.66 (C-4′), 61.64 (C-
5′), 59.89 (C-3′), 58.81 (CH2), 37.93 and 37.78 (C-2′, NCH2),
30.42 (CH2), 13.19 (CH3); CI-MS m/z 326 (M + 1)+. Anal.
(C13H19N5O5) C, H, N.
NMR (CDCl3) δ 163.47 (CO), 150.78 (CO), 136.52 (ArC), 134.76
(C-6), 128.74 (2 × ArCH), 128.32 (2 × ArCH), 127.57 (ArCH),
110.04 (C-5), 86.42 (C-1′), 84.49 (C-4′), 61.66 (C-5′), 59.86 (C-
3′), 44.41 (NCH2), 37.58 (C-2′), 13.19 (CH3); CI-MS m/z 358
(M + 1)+. Anal. (C12H16N5O4) C, H, N.
3-[(Eth yla cetoxy)m eth yl]-3′-a zid o-3′-d eoxyth ym id in e
(12j). AZT (205 mg, 0.765 mmol) NaH (25 mg, 1.04 mmol),
and ethyl bromoacetate (0.10 mL, 0.902 mmol). Purification
by silica gel flash chromatography (50% EtOAc/heptane).
12j: colorless oil (177 mg; 66%); IR (neat, cm-1) 3483, 2106,
1752, 1708, 1654, 1636; 1H NMR (CDCl3) δ 7.58 (s, H-6), 6.12
(t, J ) 6 Hz, H-1′), 4.68 (s, NCH2), 4.36 (app q, J ) 6 Hz, H-3′),
4.22 (q, J ) 7 Hz, OCH2), 3.74-3.98 (m, 3H, CH2-5′, H-4′),
3.17 (br t, OH), 2.40-2.47 (m, 2H, CH2-2′), 1.92 (s, CH3), 1.29
(t J ) 7 Hz, CH3); 13C NMR (CDCl3) δ 168.09 (CO), 162.91
(CO), 150.56 (CO), 135.12 (C-6), 110.20 (C-5), 86.76 (C-1′),
84.66 (C-4′), 61.75 (C-5′ + OCH2), 59.78 (C-3′), 42.11 (NCH2),
37.71 (CH2-2′), 14.12 (CH3), 13.13 (CH3); CI-MS m/z 354 (M +
1)+. Anal. (C14H10N5O6) C, H, N.
3-Glycid yl-3′-a zid o-3′-d eoxyth ym id in e (12l) a n d Diol
14. AZT (307 mg, 1.15 mmol), NaH (27 mg, 1.12 mmol), and
glycidyl triflate23 (347 mg, 1.68 mmol). Purification by silica
gel flash chromatography (50% EtOAc/heptane). 12l: colorless
oil (303 mg; 81%); IR (neat, cm-1) 3432, 2108, 1705, 1675, 1635;
1H NMR (CDCl3) δ 7.55 (s, H-6), 6.14 (t, J ) 6 Hz, H-1′), 4.39
1
3-(6-Hyd r oxyh exyl)-3′-a zid o-3′-d eoxyth ym id in e (12f).
AZT (546 mg, 2.04 mmol), NaH (59 mg, 2.5 mmol), and
6-bromohexanol (0.4 mL, 3.05 mmol). Purification by silica
gel flash chromatography (EtOAc). 12f: colorless oil (640 mg;
(app q, J ) 6 Hz, H-3′), 4.23 (ddd, H, J ) 14, 5, 2 Hz, NCH2),
4.07-3.75 (m, 4 H, CH2-5′, H-4′, CH2N), 3.26-3.20 (m, CH),
3.15 (br s, OH), 2.77 (t, 1H, J ) 4 Hz, cis-H of epoxide CH2),
1
2.68 (dd, H, J ) 5, 2 Hz, trans-H of epoxide CH2), 2.49-2.41
85%); IR (neat, cm-1) 3429, 2107, 1702, 1669, 1636; H NMR
(m, 2H, CH2-2′), 1.92 (s, CH3); 13C NMR (CDCl3) δ 163.35 (CO),
150.87 (CO), 134.95 (C-6), 110.19 (C-5), 86.69 (C-1′), 84.60 (C-
4′), 61.83 (C-5′), 59.96 (C-3′), 49.98 (CH), 46.45, 42.82 (CH2,
NCH2), 37.60 (C-2′), 13.18 (CH3); CI-MS m/z 324 (M + 1)+.
Anal. (C13H17N5O5) C, H, N.
1
(CDCl3) δ 7.48 (s, H-6), 6.11 (t, J ) 13 Hz, H-1′), 4.37 (app q,
J ) 6 Hz, H-3′), 4.00-3.74 (m, 5H, CH2-5′, H-4′, NCH2), 3.61
(t, 2H, J ) 6 Hz, CH2OH), 3.46 (br s, OH), 2.52-2.38 (m, 2H,
CH2-2′), 2.23 (br s, OH), 1.91 (s, CH3), 1.70-1.30 (m, 8H, CH2);
13C NMR (CDCl3) δ 163.49 (CO), 150.85 (CO), 134.63 (C-6),
110.27 (C-5), 86.85 (C-1′), 84.64 (C-4′), 62.62 (CH2OH), 61.82
(C-5′), 59.99 (C-3′), 41.24 (NCH2), 37.59 (CH2-2′), 32.50 (CH2),
27.41 (CH2), 26.50 (CH2), 25.20 (CH2), 13.23 (CH3); CI-MS m/z
368 (M + 1)+. Anal. (C16H25N5O5) C, H, N.
3-(2-Br om oeth yl)-3′-azido-3′-deoxyth ym idin e (12g). AZT
(1.07 g, 4.00 mmol), NaH (110 mg, 4.58 mmol), and 1,2-
dibromoethane (2.0 mL, 23.2 mmol). Purification by silica gel
flash chromatography (EtOAc). 12g: colorless solid (841 mg;
56%); mp 96-98 °C (CHCl3/hexane); IR (CHCl3, cm-1) 3432,
2108, 1700, 1663, 1635; 1H NMR (CDCl3) δ 7.59 (s, H-6), 6.13
(t, J ) 6 Hz, H-1′), 4.40 (app q, J ) 6 Hz, H-3′), 4.33 (t, J ) 7
Hz, NCH2), 3.95-4.00 (m, 2H, H-4′; H-5′), 3.84 (dd, 1H, J ) 9,
3 Hz, H-5′), 3.53 (t, J ) 7 Hz, CH2Br), 3.22 (br s, OH), 2.40-
2.50 (m, 2H, CH2-2′), 1.92 (s, CH3); 13C NMR (CDCl3) δ 163
(CO), 150.53 (CO), 135.10 (C-6), 110.13 (C-5), 86.69 (C-1′),
84.60 (C-4′), 61.80 (C-5′), 59.91 (C-3′), 42.09 (NCH2), 37.60 (C-
2′), 27.26 (CH2Br), 13.13 (CH3); CI-MS m/z 376 and 374 (M +
1)+. Anal. (C12H16BrN5O4) C, H, N.
3-[(Met h oxyet h oxy)m et h yl]-3′-a zid o-3′-d eoxyt h ym i-
d in e (12h ). AZT (500 mg, 1.87 mmol), NaH (54 mg, 2.25
mmol), and methoxyethoxymethyl chloride (0.32 mL, 2.80
mmol). Purification by silica gel flash chromatography (1%
MeOH/EtOAc). 12h : colorless oil (490 mg; 74%); IR (neat,
cm-1) 3467, 2119, 1702, 1676; 1H NMR (CDCl3) δ 7.42 (s, H-6),
6.08 (t, J ) 7 Hz, H-1′), 5.47 (s, OCH2N), 4.23-4.36 (m, H-3′),
4.05-3.51 (m, 7H, CH2-5′, H-4′, OCH2CH2O), 3.55 (s, CH3O),
2.59-2.33 (m, 3H, CH2-2′, OH), 1.93 (s, CH3); 13C NMR
(CDCl3) δ 163.01 (CO), 150.43 (CO), 134.87 (C-6), 109.46 (C-
5), 85.28 (C-1′), 84.18 (C-4′), 71.13 and 69.07 (OCH2CH2O),
70.59 (OCH2N), 61.14 (C-5′), 59.64 (C-3′), 58.27 (CH3O), 37.16
(CH2-2′), 12.54 (CH3); CI-MS m/z 356 (M + 1)+. Anal.
(C14H21N5O4) C, H, N.
On standing under ambient conditions, epoxide 12l slowly
added water to form diol 14: 1H NMR (CDCl3) δ 7.54 (s,
H-6), 6.12 (m, H-1′), 4.40 (app q, J ) 6 Hz, H-3′), 4.21-4.18
(m, NCH2), 4.05-3.82 (m, 4H, CH2-5′, H-4′ CHOH), 3.55 (d,
J ) 5 Hz, CH2OH), 2.68 (br s, 3H, 3 × OH), 2.54-2.44 (m,
2H, CH2-2′), 1.95 (s, CH3); 13C NMR (CDCl3) δ 164.71 (CO),
151.85 (CO), 135.26 (C-6), 110.59 (C-5), 87.01 (C-1′), 84.72 (C-
4′), 70.67 (CHOH), 63.76 (CH2OH), 61.86 (C-5′), 59.83 (C-3′),
43.90 (NCH2), 37.79 (CH2-2′), 13.29 (CH3); CI-MS m/z (CI) 342
(M + 1)+.
3-[(2,2-Dim eth yl-1,3-d ioxola n -4-yl)m eth yl]-3′-a zid o-3′-
d eoxyth ym id in e (12m ). AZT (299 mg, 1.12 mmol), NaH (38
mg, 1.58 mmol), and (2,2-dimethyl-1,3-dioxolan-4-yl)methyl
triflate (444 mg, 1.67 mmol). Purification by silica gel flash
chromatography (75% EtOAc/heptane). 12m : colorless oil
(310 mg; 73%); IR (CHCl3, cm-1) 3458, 2106, 1705, 1665, 1635;
1H NMR (neat) δ 7.33 (s, H-6), 6.03 (t, J ) 6 Hz, H-1′), 4.45-
4.29 (m, 3H), 4.08-3.75 (m, 6H), 2.64-2.34 (m, 3H, CH2-2′,
OH), 1.93 (s, CH3), 1.46 (s, CH3), 1.32 (s, CH3); 13C NMR
(CDCl3) δ 163.39 (CO), 150.72 (CO), 134.73 (C-6), 109.75 and
109.34 (C-5 and OCO), 86.25 (C-1′), 84.46 (C-4′), 73.00 (OCH),
67.54 (OCH2), 61.52 (C-5′), 59.82 (C-3′), 43.64 (NCH2), 37.50
(CH2-2′), 26.50, 25.33 (CH3), 13.05 (CH3); CI-MS m/z 382 (M
+ 1)+. Anal. (C16H23N5O6) C, H, N.
3-(3-F u r a n ylm eth yl)-3′-a zid o-3′-d eoxyth ym id in e (12o).
AZT (433 mg, 1.62 mmol), NaH (50 mg, 2.08 mmol), and
3-(bromomethyl)furan (378 mg, 2.35 mmol). Purification by
silica gel flash chromatography (50% EtOAc/heptane). 12o:
colorless oil (433 mg, 77%); IR (neat, cm-1) 3458, 2105, 1698,
1635; 1H NMR (CDCl3) δ 7.52 (s, H-6), 7.47 (d, J ) 0.9 Hz,
furan CH-O), 7.31 (t, J ) 1.4 Hz, furan CH-O), 6.48 (d, J )
1.4 Hz, furan H-4), 6.11 (t, J ) 6 Hz, H-1′), 4.93 (s, NCH2),
4.39 (dt, J ) 7, 5 Hz, H-3′), 3.94-4.01 (m, 2H, H-5′, H-4′), 3.80
(dd, 1H, J ) 12.5, 3 Hz, H-5′), 2.32-2.57 (m, 2H, CH2-2′), 1.90
(s, CH3); 13C NMR (CDCl3) δ 163.16 (C-2), 150.69 (C-4), 142.66
and 142.30 (furan ring C-2, C-5), 134.81 (C-6), 120.26 (furan
ring C-3), 111.60 (furan ring C-4), 110.40 (C-5), 87.00 (C-1′),
84.59 (C-4′), 61.92 (C-5′), 59.99 (C-3′), 37.55 (C-2′), 35.44
(NCH2), 13.22 (CH3); CI-MS m/z 348 (M + 1)+. Anal.
(C15H17N5O5) C, H, N.
3-Ben zyl-3′-a zid o-3′-d eoxyth ym id in e (12i). AZT (224
mg, 0.838 mmol), NaH (25 mg, 1.04 mmol), and benzyl bromide
(0.10 mL, 0.925 mmol). Purification by silica gel flash chro-
matography (EtOAc/heptane, 1/3). 12i: colorless needles (257
mg; 86%); mp 143-144 °C (methanol); IR (CHCl3, cm-1) 3376,
1
2113, 1701, 1670, 1654, 1628; H NMR (CDCl3) δ 7.26-7.47
(m, 6H, ArCH, H-6), 6.09 (t, J ) 6.5 Hz, H-1′), 5.10 (s, PhCH2),
4.32-4.40 (m, H-3′), 3.70-4.00 (m, 3H, CH2-5′, H-4′), 2.92-
3.00 (m, OH), 2.30-2.55 (m, 2H, CH2-2′), 1.91 (s, CH3); 13C
Meth od B: 3-(2,2-Dieth oxyeth yl)-3′-a zid o-3′-d eoxyth y-
m id in e (12n ). To a stirred solution of AZT (200 mg, 0.748

1556 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Adams et al.
mmol) in DMF (2 mL) at 40 °C under argon was added
potassium carbonate (207 mg, 1.50 mmol), potassium iodide
(37 mg, 0.22 mmol), 18-crown-6 (39 mg, 0.15 mmol), and
bromoacetaldehyde diethyl acetal (225 mL, 1.50 mmol), and
after 18 h, further potassium carbonate (207 mg, 1.50 mmol)
and bromoacetaldehyde diethyl acetal (0.225 mL, 1.50 mmol)
were added. After 48 h the reaction mixture was evaporated,
triturated with chloroform, and filtered. The filtrate was
evaporated and the residue silica gel flash column chromato-
graphed (50% EtOAc/heptane) to afford 12n as a colorless oil
(180 mg; 63%): IR (neat, cm-1) 3450, 2105, 1707, 1661; 1H
NMR (CDCl3) δ 7.55 (s, H-6), 6.07 (t, J ) 6 Hz, H-1′), 4.83 (t,
1H, J ) 6 Hz, CH(OEt)2), 4.35 (app q, J ) 5 Hz, H-3′), 4.05 (d,
J ) 5 Hz, NCH2), 3.93-3.38 (m, 10H, CH2-5′, H-4′, OH 2 ×
OCH2), 2.42-2.34 (m, 2H, CH2-2′), 1.86 (s, CH3), 1.09 (t, J )
7 Hz, 2 × CH3); 13C NMR (CDCl3) δ 163.41 (CO), 150.85 (CO),
134.84 (C-6), 109.94 (C-5), 98.59 (CH(OEt)2), 86.78 (C-1′), 84.61
(C-4′), 61.94 (2 × OCH2), 61.77 (C-5′), 60.01 (C-3′), 42.53
(NCH2), 37.62 (C-2′), 15.19 (2 × CH3), 13.20 (CH3); CI-MS m/z
384 weak (M + 1)+, 338 (M - OEt)+. Anal. (C16H25N5O6) C,
H, N.
3-[2-(5-Meth yl-2,4-oxo-2-p yr im id in -1-yl)eth yl]-3′-a zid o-
3′-d eoxyth ym id in e (12q). 5′-O-TBS AZT 9 (315 mg, 0.825
mmol), K2CO3 (318 mg, 2.30 mmol), and 1-(2-bromoethyl)-4-
methoxy-5-methylpyrimidin-2-one (208 mg, 0.842 mmol). In-
termediate 13q was separated from the excess bromide by
silica gel flash column chromatography (EtOAc) and treated
with 0.2 N HCl (20 mL) in methanol (10 mL) for 4 d and then
neutralized with Amberlite IR 68 ion-exchange resin. After
filtration and evaporation, the residue obtained was puri-
fied by flushing through a plug of silica gel (10% MeOH/
EtOAc). 12q: amorphous white solid (255 mg; 74%); mp 191-
192 °C (MeOH); IR (Nujol, cm-1) 3443, 3168, 2106, 1700, 1669,
1635; 1H NMR (DMSO-d6) δ 11.13 (br s, NH), 7.77 (s, thymine
ring H-6), 7.36 (s, thymine ring H-6), 6.04 (t, J ) 6 Hz, H-1′),
5.32 (br s, OH), 4.39 (q, J ) 6 Hz, H-3′), 3.62-4.08 (m, 7H,
N(CH2)2N, CH2-5′, H-4′), 2.29 (t, 2H, J ) 6.5 Hz, CH2-2′), 1.79
(s, CH3), 1.69 (s, CH3); 13C NMR (CDCl3) δ 164.17 and 162.59
(2 × thymine ring C-2), 151.06 and 150.27 (2 × thymine ring
C-4), 141.15 and 134.60 (2 × thymine ring C-6), 108.26 (2 ×
thymine ring C-5), 84.56 (C-1′), 84.18 (C-4′), 60.49 (C-5′), 59.69
(C-3′), 45.45 and 39.24 (N(CH2)2N), 36.54 (C-2′), 12.68 and
11.65 (2 × CH3); CI-MS m/z 420 (M + 1)+. Anal. (C17H21N7O6)
C, H, N.
3-(2-Azid oeth yl)-3′-a zid o-3′-d eoxyth ym id in e (15). To a
solution of 12g (286 mg, 0.764 mmol) in CH2Cl2 (5 mL) at room
temperature (23 °C) was added Amberlite 1R 400 (N3-) resin
(0.80 g, ca. 2 mmol of N3-). After 7 d the reaction mixture
was filtered, washing the resin well with chloroform, and the
filtrate was evaporated. The residue was flash column chro-
matographed (silica gel; 50% ethyl acetate/heptane) to afford
15 as a colorless oil (233 mg; 91%): IR (neat, cm-1) 3462, 2108,
1704, 1668, 1635; 1H NMR (CDCl3) δ 7.50 (s, H-6), 6.11 (t, J )
6.5 Hz, H-1′), 4.39 (app q, J ) 6 Hz, H-3′), 4.19 (t, J ) 6 Hz,
NCH2), 4.04-3.76 (m, 3H, H-4′, CH2-5′), 3.53 (t, J ) 6 Hz,
CH2N3), 2.92 (br s, OH), 2.52-2.40 (m, 2H, CH2-2′), 1.93 (s,
CH3); 13C NMR (CDCl3) δ 163.29 (CO), 150.82 (CO), 135.08
(C-6), 110.31 (C-5), 87.06 (C-1′), 84.63 (C-4′), 61.95 (C-5′), 59.97
(C-3′), 48.42 (CH2N3), 39.94 (CH2), 37.58 (C-2′), 13.22 (CH3);
CI-MS m/z 337 (M + 1)+. Anal. (C12H16N8O4) C, H, N.
3-(2-Br om oet h yl)-3′-a zid o-3′-d eoxy-5′-O-(ter t-b u t yld i-
m et h ylsilyl)t h ym id in e (16). To a stirred solution of 12g
(762 mg, 2.03 mmol) in DMF (10 mL) were added tert-
butyldimethylsilyl chloride (500 mg, 3.32 mmol) and imidazole
(280 mg, 4.11 mmol). After 18 h the reaction mixture was
quenched with methanol (ca. 2 mL) and the solvent removed
in vacuo after 30 min. The residue was coevaporated with
xylene and silica gel flash column chromatographed (50%
EtOAc/heptane) to afford 16 as a white solid (712 mg, 72%):
mp 92-93 °C; IR (CHCl3, cm-1) 2106, 1706, 1668, 1652; 1H
NMR (CDCl3) δ 7.44 (s, H-6), 6.22 (t, J ) 7 Hz, H-1′), 4.33 (t,
J ) 7 Hz, CH2), 4.26-4.20 (m, H-3′) 3.98-3.97 (m, 2H, H-5′,
H-4′), 3.79 (dd, 1H, J ) 11, 2 Hz, H-5′), 3.53 (t, 2H, J ) 7 Hz,
CH2Br), 2.45 (ddd, 1H, J ) 14, 6, 5 Hz, H-2′), 2.22 (dt 1H, J )
14, 7 Hz, H-2′), 1.92 (s, CH3), 0.92 (s, 9H, CH3), 0.12 (s, 6H,
(CH3)2Si); 13C NMR (CDCl3) δ 163.02 (CO), 150.62 (CO), 133.52
(C-6), 110.26 (C-5), 85.37 (C-1′), 84.63 (C-4′), 62.94 (C-5′), 60.48
(C-3′), 42.20 (CH2), 38.17 (C-2′), 27.35 (CH2Br), 26.01 (CH3),
18.47 (CSi), 13.32 (CH3), -5.26 (CH3Si); CI-MS m/z 490 and
488 (M + 1)+. Anal. (C18H30N5O4SiBr) C, H, N.
Meth od C: 3-P en tyl-3′-a zid o-3′-d eoxyth ym id in e (12b).
5′-O-TBS AZT 9 (187 mg, 0.490 mmol), K2CO3 (168 mg, 1.22
mmol), and pentyl bromide (0.150 mL, 1.21 mmol). Intermedi-
ate 13b was directly treated with HF in CH3CN, and the
residue obtained after workup was purified by silica gel flash
column chromatography (50% EtOAc/heptane). 12b: colorless
oil (159 mg; 96%); IR (CHCl3, cm-1) 3449, 2105, 1702, 1670,
1
1636; H NMR (neat) δ 7.55 (s, H-6), 6.13 (t, J ) 6 Hz, H-1′),
4.41 (app, q, J ) 6 Hz, H-3′), 3.78-4.03 (m, 5H, H-4′, CH2-5′,
NCH2), 3.49 (t, OH), 2.39-2.54 (m, 2H, CH2-2′), 1.91 (s, CH3),
1.58 (p, 2H, J ) 7 Hz, CH2), 1.24-1.37 (m, 4H, 2 × CH2), 0.87
(t, 3H, J ) 7 Hz, CH3); 13C NMR (CDCl3) δ 163.49 (CO), 150.77
(CO), 134.61 (C-6), 110.16 (C-5), 86.79 (C-1′), 84.60 (C-4′), 61.84
(C-5′), 59.98 (C-3′), 41.45 (NCH2), 37.59 (C-2′), 29.04, 27.20,
22.32 (3 × CH2), 13.92 (CH3), 13.24 (CH3); CI-MS m/z 338 (M
+ 1)+. Anal. (C15H23N5O4) C, H, N.
3-[(Dieth ylp h osp h on o)m eth yl]-3′-a zid o-3′-d eoxyth ym i-
d in e (12k ). 5′-O-TBS AZT 9 (343 mg, 0.898 mmol), K2CO3
(255 mg, 1.84 mmol), and (diethylphosphono)methyl triflate
(411 mg, 1.34 mmol). Intermediate 13k was directly treated
with tetrabutylammonium fluoride (1.0 M in THF; 0.90 mL,
0.90 mmol) and the residue obtained after workup was purified
by silica gel flash column chromatography (EtOAc). 12k :
colorless oil (203 mg; 54%). IR (neat, cm-1) 3361, 2104, 1708,
1654; 1H NMR (CDCl3) δ 7.67 (s, H-6), 6.14 (t, J ) 6 Hz, H-1′),
4.12-4.43 (m, 8H, H-3′, NCH2, OH, 2 × OCH2), 3.99 (br d,
1H, J ) 12 Hz, H-5′), 3.88-3.91 (m, H-4′), 3.71 (br d, 1H, J )
12 Hz, H-5′), 2.26-2.42 (m, 2H, CH2-2′), 1.88 (s, CH3), 1.36 (t,
J ) 7 Hz, CH3), 1.35 (t, J ) 7 Hz, CH3); 13C NMR (CDCl3) δ
162.63 (CO), 150.28 (CO), 134.63 (C-6), 109.89 (C-5), 86.33 (C-
1′), 84.96 (C-4′), 63.99 (d, J (13C-31P) ) 6 Hz, P(O)OCH2), 62.78
(d, J (13C-31P) ) 6 Hz, P(O)OCH2), 61.24 (C-5′), 59.70 (C-3′),
38.18 (C-2′), 36.43 (d, J (13C-31P) ) 156 Hz, NCH2P(O)), 16.35
(d, J (13C-31P) ) 6 Hz, P(O)(OCH2CH3)2), 13.03 (CH3); CI-MS
m/z 418 (M + 1)+. Anal. (C15H24N5O7P) C, H, N.
3-[2-(4-Meth oxy-5-m eth yl-2-oxo-2-pyr im idin -1-yl)eth yl]-
3′-a zid o-3′-d eoxyth ym id in e (12p ). 5′-O-TBS AZT 9 (145
mg, 0.380 mmol), K2CO3 (143 mg, 1.03 mmol), and 1-(2-
bromoethyl)-4-methoxy-5-methylpyrimidin-2-one (123 mg, 0.498
mmol). Intermediate 13p was directly treated with tetrabu-
tylammonium fluoride (1.0 M in THF; 0.40 mL, 0.40 mmol),
and the residue obtained after workup was purified by silica
gel flash column chromatography (5% MeOH/EtOAc). 12p :
amorphous white solid (158 mg; 96%); mp 126-128 °C (CHCl3/
hexane); IR (Nujol, cm-1) 3244, 2090, 1698, 1665, 1636; 1H
NMR (CDCl3) δ 7.73 (s, H-6′′), 7.21 (s, H-6), 6.10 (t, J ) 6 Hz,
H-1′), 4.46 (q, J ) 5 Hz, H-3′), 3.84-4.30 (m, 8H, N(CH2)2N,
CH2-5′, H-4′, OH), 3.94 (s, OCH3), 2.35-2.47 (m, 2H, CH2-2′),
1.92 (s, CH3′′), 1.87 (s, CH3); 13C NMR (CDCl3) δ 170.85 (C-
2′′), 163.41 (C-2), 157.17 (C-4′′), 150.83 (C-4), 144.09 (C-6′′),
135.13 (C-6), 109.63 and 105.04 (2 × thymidine ring C-5), 86.51
(C-1′), 85.11 (C-4′), 61.56 (C-5′), 60.12 (C-3′), 54.43 (OCH3),
47.53 and 39.94 (N(CH2)2N), 37.65 (C-2′), 13.10 and 11.94 (2
× CH3); CI-MS m/z 434 (M + 1)+. Anal. (C18H23N7O6) C, H,
N.
3-(2-P h th alim idoeth yl)-3′-azido-3′-deoxyth ym idin e (17).
To a stirred solution of phthalimide potassium salt (113 mg,
0.607 mmol) and 18-crown-6 (38.5 mg, 0.146 mmol) in DMF
(3 mL) at 75 °C was added 16 (275 mg, 0.563 mmol). After 18
h the solvent was removed in vacuo, and the residue was
coevaporated with xylene and then silica gel flash column
chromatographed (50% ethyl acetate/heptane) to afford a
colorless oil (275 mg), which was directly treated with 1:9 40%
HF in CH3CN (2 mL). After 1.5 h the reaction mixture was
evaporated to dryness, and the residue was flash column
chromatographed (silica gel; 50% ethyl acetate/heptane) to
afford 17 as a colorless oil (204 mg; 82%): IR (neat, cm-1) 3472,
2105, 1717, 1669, 1637; 1H NMR (CDCl3) δ 7.83-7.68 (m, 4H,
Ar-H), 7.30 (d, J ) 1 Hz, H-6), 5.86 (dd, J ) 7, 5 Hz, H-1′),
4.37-4.25 (m, 3H, H-3′, CH2N), 4.05-3.67 (m, 5H, CH2-5′,
CH2N, H-4′), 2.81 (br m, OH), 2.47 (ddd, 1H, J ) 13, 7, 5 Hz,
H-2′), 2.28 (dt, 1H, J ) 13, 6 Hz, H-2′), 1.81 (d, J ) 1 Hz, CH3);

N-3-Substituted Thymidine Nucleoside Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1557
13C NMR (CDCl3) δ 168.33 (2 × CO), 163.46 (CO), 150.83 (CO),
135.02 (C-5), 133.91 (2 × ArCH), 131.89 (2 × ArC), 123.06 (2
× ArCH), 109.64 (C-5), 87.02 (C-1′), 84.63 (C-4′), 61.45 (C-5′),
59.43 (C-3′), 39.74 (CH2), 37.36 (CH2), 35.88 (CH2), 12.99 (CH3);
CI-MS m/z 441 (M + 1)+. Anal. (C20H20N6O6) C, H, N.
studies, all derivatives were dissolved in DMSO. The same
final concentration of DMSO was used in the control experi-
ments.
An tivir a l Assa y on CEM-c113 Cells a n d RT Dosa ge.
The antiviral assay on CEM c113 cells were performed as
follows.15 Briefly, 1 × 104 cells/wells were incubated in
microtitration plates for 1 h with 10-fold diluted concentrations
of the compound then infected by HIV-1 LAI strain. Cultures
were incubated for 7 days at 37 °C in a CO2 incubator (5%).
At day 7 the supernatant was removed for reverse tran-
scriptase (RT) assay to determine inhibition of viral production
(EC50RT), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide was added in cell suspension to determine cell
viability (CC50 and EC50MTT) by the MTT assay. The toxicity
of the products (CC50) was defined in relation to the viability
of uninfected untreated control cells. The inhibition of CPE
(EC50MTT) of the products was defined in relation to the infected
untreated cells.
3-[(3′′′-Azid o-3′′′-d eoxyth ym id in -3′′-yl)eth yl]-3′-a zid o-3′-
d eoxyth ym id in e (18). To a vigorously stirred solution of 5′-
O-TBS 9 (102 mg, 0.266 mmol) in dry DMF (2 mL) was added
activated 4 Å sieve powder, powdered potassium carbonate (94
mg, 0.68 mmol), and bromide 16 (165 mg, 0.338 mmol). After
24 h the reaction mixture was filtered, washing well with
chloroform, the filtrate was evaporated, and the residue was
flash column chromatographed (silica gel; 25% ethyl acetate/
heptane) to remove the excess 16 and afford the silylated
product as a viscous oil (163 mg). The oil was treated for 1 h
with 40% HF (0.30 mL) in CH3CN (3 mL), evaporated, and
flash column chromatographed (silica gel; EtOAc) to afford the
deprotected 18 as a white solid (102 mg; 68%): IR (CHCl3,
1
cm-1) 3450, 2106, 1701, 1670, 1636; H NMR (CDCl3) δ 7.31
(s, 2 × H-6), 5.88 (t, J ) 6 Hz, 2 × H-1′), 4.35 (q, J ) 6 Hz, 2
× H-3′), 4.23 (s, 4H, NCH2CH2N), 4.84-4.94 (m, 4H, 2 × H-4′,
2 × H-5′), 3.71 (dd, 2H, J ) 12.5, 3 Hz, 2 × H-5′), 3.57 (br s,
2 × OH), 2.55 (ddd, 2H, J ) 13, 7.5, 5.5 Hz, 2 × H-2′), 2.34
(dt, 2H, J ) 13, 6.5 Hz, 2 × H-2′), 1.82 (s, 2 × CH3); 13C NMR
(CDCl3) (all peaks × 2) δ 163.75 (CO), 151.07 (CO) 135.51 (C-
6), 109.75 (C-5), 81.11 (C-1′), 84.87 (C-4′), 61.75 (C-5′), 59.77
(C-3′), 39.35 (NCH2CH2N), 37.10 (C-2′), 13.14 (CH3); CI-MS
m/z 561 (M + 1)+. Anal. (C22H28N10O8) C, H, N.
Ack n ow led gm en t. This work has been generously
supported by the French Agency for Research Against
AIDS (ANRS), the Association de Recherches Contre le
Cancer (ARC), the University of Bordeaux II, the
Conseil Re´gional d’Aquitaine, and the Rhoˆne-Poulenc
Rorer Company.
Refer en ces
3-[[3′′-(Hyd r oxym eth yl)isoxa zol-5′′-yl]m eth yl]-3′-a zid o-
3′-d eoxyth ym id in e (19). To a stirred of 12c (295 mg, 0.96
mmol) in DMF (10 mL) was added tert-butyldimethylsilyl
chloride (217 mg, 1.44 mmol) and imidazole (105 mg, 1.54
mmol). After 16 h the reaction mixture was quenched with
methanol (ca. 5 mL) and the solvent removed in vacuo after
30 min. The residue was coevaporated with xylene and silica
gel flash column chromatographed (50% EtOAc/heptane) to
afford an oil (263 mg; 65%): 1H NMR (CDCl3) δ 7.45 (s, H-6),
6.25 (t, J ) 6.5 Hz, H-1′), 5.78-5.98 (m, CHdCH2), 5.16-5.30
(m, CHdCH2), 4.55 (d, J ) 5.5 Hz, NCH2), 4.20-4.27 (m, H-3′),
3.77-3.98 (m, 3H, CH2-5′, H-4′), 2.40-2.52 (m, 1H, H-2′),
2.16-2.30 (m, 1H, H-2′), 1.94 (s, CH3), 0.93 (s, 9H, (CH3)3CSi),
0.13 (s, 6H, (CH3)2Si).
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To a solution of this intermediate in toluene (50 °C) was
added triethylamine (25 µL, 0.18 mmol), 2-nitroethyl tert-
butyldimethylsilyl ether (62 mg, 0.30 mmol), and phenyl
isocyanate (0.37 mL, 3.40 mmol). After 3 d the reaction
mixture was filtered, evaporated, and treated with 1:9 40%
HF in CH3CN (0.06 mL). After 1 h the reaction mixture was
evaporated to dryness, and the residue was flash column
chromatographed (silica gel; ethyl acetate/methanol 2%) to
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1
2107, 1669, 1636; H NMR (CDCl3) δ 7.50 (s, H-6), 6.10 (td,
1H, J ) 6 Hz, NCH2), 4.02-4.42 (m, 6H, CH2-5′, H-4′, H-3′,
CH2OH), 3.49 (s, 2 × OH), 3.12 (dd, 1H, J ) 17, 10 Hz, CH2
isoxazoline), 2.90 (dd, 1H, J ) 17, 5.5 Hz, CH2 isoxazoline),
2.32-2.58 (m, 2H, CH2-2′), 1.89 (s, CH3); 13C NMR (CDCl3) δ
163.30 (CO), 159.08 (CdNO), 151.29 (CO), 135.22 (C-6), 110.33
(C-5), 86.83 (C-1′), 84.67 (C-4′), 76.80 (CHO isoxazoline), 61.82
(C-5′), 59.96 (C-3′), 57.90 (CH2OH), (43.64 (NCH2), 38.88-37.56
(CH2′, CH2 isoxazoline), 13.22 (CH3); CI-MS m/z 381 (M + 1)+.
Anal. (C15H20N6O6) C, H, N.
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Rever se Tr a n scr ip ta se Assa y. As previously described,20
yeast cells which have been transformed with the vector pAB
24/RT-4 were used to purify the recombinant HIV-1 RT
employed in the bioassays. The reaction mixture contained
in a final volume of 0.05 mL, 50 mM Tris-HCl, pH 8.0, 5 mM
MgCl2, 4 mM dithiothreitol, 0.48 A260/mL of poly(A)-oligo(dT)
(5:1), 0.5 µCi [3H]dTTP (46 Ci/mmol), 20 µM dTTP, 80 mM
KCl, 1 µg of bovine serum albumin, and 20-50 nM RT.
Incubation was carried out at 37 °C for 10 min.
The reactions were stopped by the addition of 1 mL of cold
10% trichloroacetic acid plus 0.1 M sodium pyrophosphate. The
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PPO/POPOP/toluene scintillation mixture. For the inhibition
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J M9600095