Uncharged AZT and D4T derivatives of phosphonoformic and phosphonoacetic acids as anti-HIV pronucleosides

Article abstract of DOI:10.1021/jm0310176

Two series of new lipophilic phosphonoformate and phosphonoacetate derivatives of AZT and d4T were synthesized and evaluated as anti-HIV agents. The efficacy of some of the synthesized compounds in cell cultures infected with HIV-1 was higher than that of

Full text of DOI:10.1021/jm0310176

3
606
J . Med. Chem. 2004, 47, 3606-3614
Un ch a r ged AZT a n d D4T Der iva tives of P h osp h on ofor m ic a n d P h osp h on oa cetic
Acid s a s An ti-HIV P r on u cleosid es
,
†
†
†
†
Elena A. Shirokova,* Maxim V. J asko, Anastasiya L. Khandazhinskaya, Alexander V. Ivanov,
†
†
†
‡
§
Dmitry V. Yanvarev, Yury S. Skoblov, Vladimir A. Mitkevich, Eduard V. Bocharov, Tatyana R. Pronyaeva,
§
†
§
Nina V. Fedyuk, Marina K. Kukhanova, and Andrey G. Pokrovsky
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str., Moscow 119991, Russian Federation;
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/ 10 Miklukho-Maklaya str.,
Moscow 117997, Russian Federation; and State Research Center of Virology & Biotechnology “Vector”, Koltsovo, Novosibirsk
region 633159, Russian Federation
Received September 2, 2003
Two series of new lipophilic phosphonoformate and phosphonoacetate derivatives of AZT and
d4T were synthesized and evaluated as anti-HIV agents. The efficacy of some of the synthesized
compounds in cell cultures infected with HIV-1 was higher than that of the parent nucleosides
and only slightly correlated to their stability in the phosphate buffer and human blood serum.
The synthesized phosphonates are most probably prodrug forms of the corresponding nucleo-
sides.
In tr od u ction
Modified nucleosides used in medical practice for
AIDS therapy are targeted at the inhibition of the
proviral DNA synthesis catalyzed by HIV reverse tran-
scriptase. To become active, these nucleosides must be
phosphorylated by cellular kinases to give successively
the corresponding 5′-mono-, -di-, and -triphosphates.
The efficacy of this process is extremely low (for
example, 0.3% for AZT). Therefore, many efforts have
been made to improve therapeutic properties by short-
ening this cascade and bypassing at least the first
phosphorylation step. This approach has resulted in the
1
-8
synthesis of numerous nucleotide analogues.
Another
way of increasing the efficacy of the currently used
drugs involves altering the bioavailability of the drug
itself. For example, different modifications have been
performed in the AZT molecule with the goal to improve
its pharmacological properties (refs 9, 10, and references
therein).
On the basis of our ongoing studies of antiviral
nucleoside analogues, we describe herein the synthesis
and chemical and enzymatic stability of several new
AZT and d4T derivatives containing uncharged 5′-
(alkyl)phosphonoformate and 5′-(alkyl)phosphonoace-
tate residues (1-8) as well as their evaluation as
potential anti-HIV agents in MT-4 cell cultures infected
with HIV-1.
Resu lts
Ch em istr y. The target triesters 1, 2, 3a , and 4a were
synthesized by the reaction of the corresponding nucleo-
side with ethyl ethoxycarbonylphosphonyl chloride or
ethyl ethoxycarbonylmethylphosphonyl chloride, respec-
tively, obtained from commercially available triesters.11,12
The product yields did not exceed 60-70% (in particular,
because of the formation of 5′-chloro-5′-deoxy deriva-
tives).
A general scheme of synthesis for the target triesters
and amidodiesters of types 1-8 involved the preparation
of the corresponding charged derivatives 9-20 and their
*
To whom correspondence should be addressed. Phone: 7 (095) 135-
2
255; fax: 7 (095) 135-1405; e-mail: shirokova-elena@list.ru.
†
Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences.
‡
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Rus-
sian Academy of Sciences.
§
State Research Center of Virology & Biotechnology “Vector”.
1
0.1021/jm0310176 CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/29/2004

Anti-HIV Pronucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3607
Sch em e 1
a
(i) RNH2; (ii) TPSCl, Py, DMF, ROH; (iii) DEAD, PPh3, ROH.
subsequent esterification (Scheme 1). Phosphonofor-
mates 9 and 10 and phosphonoacetates 11 and 12 were
obtained by the coupling of the corresponding nucleoside
with ethoxycarbonylphosphonic or ethoxycarbonyl-
methylphosphonic acids in the presence of 1,3-dicyclo-
hexylcarbodiimide (DCC).13 The yields of products
achieved 70-80%. Phosphonates 9-12 were converted
to the corresponding amides 13-20 by treatment with
the corresponding amines. Phosphonates 9-20 were
esterified by the coupling with the corresponding alco-
hols either in the presence of 2,4,6-triisopropylben-
NMR spectral patterns were different: one, a singlet
at 7.24 ppm, while the other was a doublet at 6.83 ppm
with J P-H 35 Hz. Using HPLC on a reverse-phase
3
Nucleosil C-18 column in the elution system (a) (see the
Experimental Section) we could easily separate the
diastereomers of 5a (their retention times differed by
more than 2 min and were 13.3 and 15.4 min, respec-
3
1
tively). The P NMR spectra supported the hypothesis
of an interaction between the phosphorus atom and one
of the amide protons (see below).
According to the spatial model of compound 5a
(constructed with the HYPERCHEM program), the
ribose ring of the R-isomer is in close proximity to the
1
4
zenesulfonyl chloride (TPSCl) or under the Mitsunobu
reaction conditions.15 One disadvantage to the first
procedure is the requisite of use of a large excess of the
condensing reagent (2-3 equiv) because of the side
formation of the corresponding TPSOR esters. In con-
trast, however, the Mitsunobu reaction is attractive for
the preparation of pH-sensitive compounds, since it
proceeds under neutral conditions, although its efficacy
noticeably falls in the case of application of secondary
1
5
alcohols.
The newly formed chiral center at the phosphorus
ethyl group of the chiral phosphorus atom, while in the
S-isomer it is neighbored to the amide group. As a
result, we then tried to establish both an absolute
configuration for the 5a -isomers and to elucidate the
exact nature of the factors affecting their chromato-
graphic properties.
atom in compounds 1-8 results in the formation of two
3
1
diastereoisomers. Indeed, two similar P-signals in the
ratio of approximately 1:1 were observed for each of
3
1
these phosphonates in their P NMR spectra acquired
1
with H broad-band decoupling (see Experimental Sec-
1
tion). The 1D H NMR spectrum of compound 5a
The CD spectra of AZT, A and B isomers of amide
dissolved in H2O also supported the existence of two
diastereoisomeric forms. The resonances of the ethyl and
amino groups were doubled with the same intensity as
the resonances in the 31P spectral pattern (data are not
shown).
5
2
a , and phosphonate 13 in the wavelength range of
20-370 nm are presented in Figure 1a. A positive
maximum on CD curves in the region of 275-285 nm
demonstrates the prevalence of the anti-conformation
of the thymine base in these compounds. The differ-
16
It is noteworthy that splitting patterns for the protons
ences in their CD spectra can be explained by a different
ratio of syn and anti conformations of the pyrimidine
1
of the NH2CO group of compound 5a observed in
H

3
608 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Shirokova et al.
F igu r e 1. (a) CD spectra of AZT (dotted line), A (solid line),
and B (dot-and-dashed line) isomers ofamide 5a and ami-
nocarbonylphosphonate 13 (dashed line) in the far UV region
2 4
at pH 6.5 (5 mM NaH PO , 25 °C). (b) CD spectra in ethanol.
base. These conformations are dependent on the various
conformations of the furanose ring, which result from
the presence of different substituents at the 5′ C atom.
F igu r e 2. 1H NMR spectra of compound 5a . (a) 1H NMR
To further explore our hypothesis regarding the effect
of substituents at the phosphorus atom on the confor-
mation of the compounds under study, we compared
their CD spectra in ethanol in order to disturb the
interactions that would occur in water. Figure 1b shows
that all four curves were nearly identical. This allowed
us to postulate that the difference between phospho-
nates A and B was based on the difference in the
configuration of the substituents at the phosphorus
atom.
spectrum in 90% H
2
5
2
O and 10% D
D H- H ROESY spectrum (mixing time 200 ms, 99.9% D
°C) cut on proton H-6 frequency. The peak assignments are
2
O at 5 °C; (b) 1D section of
1
1
2
O,
marked. Resonances of the A and B isomers are labeled as A
and B, respectively.
Ta ble 1. Hydrolysis of the Synthesized Phosphonates in
Phosphate Buffer (pH 7.2, 20 °C)
AZT derivative
half-life
d4T derivative
half-life
1
3b
5
5
5
7
10 min ( 2
.8 h
2.5 h ( 0.25
6 h ( 0.30
10 h ( 0.30
.20 h
2
30 min ( 3
.8 h
1
4a
6a
6b
6c
8
The 1D H NMR spectrum of compound 5a dissolved
a
b
c
7 h ( 0.30
15 h ( 0.33
>20 h
in H2O (Figure 2a) also supported the existence of
stereoisomers [the resonances of the protons that dis-
play the NOE interaction with the thymine H-6 proton
owing to their spatial proximity (<5 Å) are shown]. The
analysis of the distances between the H-6 proton and
the 1′-5′ protons of the ribose ring confirmed the
prevalence of the base anti-conformation in the phos-
phonate 5a and implied the absence of hydrophobic
interactions between the thymine ring and the ethoxy
group at the phosphorus atom.
.24 h
a more perfectly packed hydrophobic “core”. This may
explain the difference in the chromatographic behavior
of the diastereoisomers A and B.
Ch em ica l Sta bility. Chemical stability of the syn-
thesized compounds was studied in the phosphate buffer
(pH 7.2) at 20 °C and the concentration of the compound
Analysis of the 1D section of 2D 1H- H ROESY
spectrum of compound 5a showed a weak NOE interac-
tion between the ribose protons and the substituents
at the phosphorus atom (Figure 2b). Moreover, for
isomer A, in contrast to B, we observed NOE contacts
between the ethyl CH2 protons and the 3′- and 4′-
protons of the ribose ring. This suggested a spatial
proximity of the ethyl group and the ribose ring in
isomer A. On the basis of all evidence obtained, the R
configuration can be assigned to isomer A and the S
configuration, to isomer B.
1
under study of 0.5 mM (Table 1). The composition of
the reaction mixture was analyzed by HPLC on an RP-
1
8 column.
In the case of phosphonoformate triesters 1 and 2 we
observed the formation of two products, a nucleoside and
the corresponding 5′-ethyl phosphite of AZT or d4T,
respectively, (ROP(H)(O)O-nucleoside), although the
nucleoside was formed considerably faster. The nucleo-
side was a major product; indeed, the amount of the
minor product at the point of 50% hydrolysis of the
starting triester did not exceed 10-12% of the total
amount of products. Conversely, the phosphonoacetate
derivatives of both AZT and d4T (3, 4, 7, and 8) were
very stable: after 8 h under the same conditions we
observed more than 95% of the phosphonates under
study and only 3-4% of the corresponding nucleoside.
In contrast, the hydrolysis of amides 5 and 6 each
yielded one major product, which was identified as the
corresponding nucleoside AZT or d4T. We performed a
Additionally, the 1H- H ROESY spectral pattern
1
3
showed that a large J H-P coupling constant of 35 Hz
for one of the protons of amide group can result from
the NHtrans proton located in the trans position relative
to the phosphorus atom. Hence, the NHtrans is located
farther from the ribose ring than the amide cis proton
NHcis. It follows, therefore, that the ethyl group in
isomer A is located closer to the thymine base and favors

Anti-HIV Pronucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3609
F igu r e 3. The time-pH dependence of AZT 5′-(ethyl)ami-
nocarbonylphosphonate 5a hydrolysis.
F igu r e 4. Hydrolysis of AZT and d4T 5′-(ethyl)aminocarbo-
nylphosphonates 5a and 6a at 37 °C. Curves I and II, 5a and
6a , respectively, in human blood serum; III and IV, 5a and
more detailed study of the composition of the reaction
mixture at the hydrolysis of compound 5a at 37 °C and
pH 7.2. In the course of the analysis we observed two
products, one of them being formed much more quickly.
AZT and phosphonate 13 were found to be major and
minor products, respectively, and the amount of the
latter did not exceed 10% of the total amount of all the
components. To identify the hydrolysis products, au-
thentic AZT and AZT 5′-aminocarbonylphosphonate 13
were used for comparison.
6
a , respectively, in phosphate buffer (pH 7.2).
Ta ble 2. Hydrolysis of the Phosphonates in Human Blood
Serum (37 °C)
AZT derivative
half-life
d4T derivative
half-life
5
7
a
7 min ( 2
>20 h
6a
8
21 min ( 3
>24 h
to their ability to suppress viral replication by measur-
ing the amount of viral antigen p24.
We also studied the chemical stability of 0.45 mM
amide 5a in the 0.05 M phosphate buffer at 37 °C in
the pH range of 5.5-7.8. The time dependence of its
hydrolysis relative to pH is presented in Figure 3; the
stability of compound 5a is obviously strongly affected
by the pH. For example, the half-life at pH 5.5 was
about 16 h, whereas at pH 7.8 it was reduced to 34 min.
This implies that the hydrolysis rate of amide 5a was
grew dramatically with a pH increase and achieved
maximum values at pH > 7.5. The plotted kinetic
hydrolysis curve is inherent for first-order reactions. The
half-life of the amide at pH 7.2 and 37 °C was about 50
min.
The phosphonoacetate derivatives of both series (3,
4, 7, and 8) were inactive; their toxicities were compa-
rable with those of parent nucleosides, except triesters
4, which were twice as toxic as compared with d4T. The
aminocarbonyl derivatives of both series displayed a
high anti-HIV effect. This effect was more pronounced
in the case of AZT compounds (5a -c). For the corre-
sponding d4T counterparts, the gain was more moder-
ate, since a higher activity was accompanied by a higher
toxicity. It is also worth mentioning that AZT phospho-
nates 1 and 5a -c inhibited the virus reprlication by
9
0% at the concentrations, which were considerably
lower than those for AZT. Anti-HIV properties of
homogeneous (R)-5a and (S)-5a coincided with those for
the diastereomeric mixture (data not shown).
Similar experiments were carried out with A and B
isomers of amide 5a . The hydrolytic properties of
individual isomers completely coincided with those of
the diastereomeric mixture 5a (data not shown).
Discu ssion
En zym a tic Hyd r olysis. We also studied enzymatic
stability of amides 5-8. In experiments with human
blood serum, which is enriched with hydrolyzing en-
zymes, our results showed that phosphonoformate
derivatives 5 and 6 were readily cleaved to give the
corresponding nucleosides (Figure 4), whereas phospho-
noacetate derivatives 7 and 8 proved to be stable under
the same conditions (Table 2).
The insight into the nature of viral diseases and the
discovery of valid targets for the antiviral agents have
enabled the development of a number of drugs for the
treatment of HIV, herpes simplex viruses, cytomega-
lovirus, and other infections. One focus for further
studies has been to increase the efficacy of the approved
drugs. A chemical approach has involved structural
modifications that could potentially improve the bio-
availability of these molecules by affecting their pen-
etration through cell membranes, as well as the rate of
transformation into the active form (i.e. metabolism).
The enzymatic hydrolysis of amide 5a in the presence
of hydrolases, such as snake venom phosphodiesterase,
calf spleen phosphodiesterase, and P1 endonuclease,
was not observed (data not shown). On the contrary, in
the presence of Crotalus atrox snake venom preparation,
amide 5a was rapidly cleaved to give AZT, with a half-
life less than 5 min. The properties of homogeneous (R)-
In the early 1990s, AZT-phosphonoformic acid con-
jugates of type 21 were prepared with the expectation
that both fragments being biologically active can display
1
1,17
5
a and (S)-5a diastereomers in these experiments did
synergism.
These diesters showed some advantages
not differ from those of the diastereomeric mixture (data
not shown).
in the cultures resistant to both phosphonoformic acid
and AZT, although they exhibited lower anti-HIV
activities and (as a rule) selectivity indexes than AZT
An tivir a l Activity. Antiviral activity of the synthe-
sized compounds was studied in MT-4 cell cultures
infected with HIV-1, strain BIII (Table 3). The activity
of the compounds under study was evaluated according
1
7
against the wild-type strain.
Later Meier et al. reported the synthesis and antiviral
activity of compounds of type 22, which are highly

3
610 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Shirokova et al.
Ta ble 3. Antiviral Activity of the Compounds under Study
a
b
Compound concentration required to cause a 50% inhibition of cell proliferation. Compound concentration required to cause a 50%
c
d
inhibition of HIV replication. Compound concentration required to cause a 90% inhibition of HIV replication. CD50/ID50.
lipophilic.18 Triesters of type 22 showed rather good
activity in cell cultures infected with HIV-1 but were
inactive in the thymidine kinase-deficient cells. In
addition, the stability of these compounds was low. We
believed that a more detailed investigation of the
structures of this type with varied substituents both at
the phosphorus atom and carbonyl group could prove
interesting.

Anti-HIV Pronucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3611
displayed reasonable stability and good anti-HIV po-
tency in vitro and are worth further investigation.
Exp er im en ta l Section
Bromotrimethylsilane, diethyl azodicarboxylate, triphen-
ylphosphine, triethylphosphonoformate, and triethylphospho-
noacetate were from Fluka; TPSCl, pyridine, and DMF were
from Aldrich. AZT was a kind gift of “AZT Association” Ltd
It is worth mentioning that the synthesized d4T
derivatives were more stable in comparison with the
corresponding AZT phosphonates (Tables 1 and 2). All
the prepared aminocarbonylphosphonates 5-8 exhib-
ited higher stability than the corresponding triesters
(Moscow, Russia).
Column chromatography was performed on silica gel 60
40-63 µm), reverse-phase chromatography was carried out
(
on LiChroprep RP-8 and LiChroprep RP-18 (25-40 µm)
(
Merck), ion-exchange column chromatography was performed
-
1
-4 both in the buffer and in human blood serum. In
on DEAE-Toyopearl (HCO3 ) (Toyosoda, J apan), and HPLC
was performed on a Gilson chromatograph (France) supplied
with an LKB 2220 integrator. A Nucleosil 100 C-18 (5 µm)
column (4 × 150 mm) and two eluting systems were used. (a)
both series phosphonoacetate derivatives 3, 4, 7, and 8
were considerably more stable than phosphonoformates
1
, 2, 5, and 6.
2 4
Buffer A: 0.065 M NaH PO ; buffer B: 72% ethanol; gradi-
It should be noted that phosphonoformate and
ent: 0-5 min, 0% B; 5-10 min, 0-15% B; 10-30 min, 15-
phosphonoacetate derivatives were hydrolyzed differ-
ently. Particularly, in the case of amides 5 and 6, the
major mechanism involved the degradation of the phos-
phodiester bond while releasing nucleoside. In the case
of triesters 1 and 2, however, we observed the formation
of P-esters of AZT or d4T 5′-H-phosphonates in addition
to the corresponding nucleoside. Similar transforma-
tions of phosphonoformate esters into H-phosphonate
esters were reported in ref 19.
2
5% B; 30-35 min, 25-100% B. The flow rate was 0.5 mL/
min. (b) Buffer A: 50 mM TEAB; buffer B: 70% ethanol;
gradient: 0-5 min, 0% B; 5-30 min, 0-30% B; 30-40 min,
3
0-32% B; 40-45 min, 32-100% B. The flow rate was 0.5
mL/min.
NMR spectra were registered on an AMX III-400 spectrom-
1
eter (Bruker) with the working frequency of 400 MHz for
Me Si as an internal standard for organic solvents and sodium
-(trimethylsilyl)-1-propanesulfonate for D O) and 162 MHz
for P NMR (with phosphorus-proton interaction decoupling,
H
(
4
3
2
3
1
1
31
According to the hydrolysis studies, the resulting
phosphonoformates are depot forms of the corresponding
nucleosides that are actual anti-HIV drugs. To elucidate
potential enzymatic pathways of the conversion of the
synthesized phosphonates into antiviral nucleosides, we
tested amide 5a in several enzymatic systems. Exonu-
cleases (snake venom and calf spleen phosphodiesteras-
es) as well as Penicillinium citrum endonuclease P1 did
not digest amide 5a . At the same time, Crotalus atrox
snake venom preparation efficiently hydrolyzed com-
pound 5a to give AZT. Most probably, this effect can be
attributed to the action of 5′-nucleotidase, which is
3 4
85% H PO as an external standard). 1D ( H and P) and 2D
(
6
1
1
H- H) NMR experiments were performed on a Varian Unity-
00 spectrometer with 0.6 mL of 5 mM water solution (99.9%
D
5
2
O or 90% H
2
O + 10% D
2
O) of diastereomeric mixture 5a at
°C and 30 °C. The homonuclear 2D NMR spectra were
21
acquired at 5 °C using pulsed-field-gradient watergate
scheme for water signal suppression: TOCSY (2) with isotropic
mixing period of 80 ms and ROESY with mixing time 200
2
2
1
2
ms. The 1D H spectra of 0.5 mM water solution (99.9% D O)
of individual A and B isomers of compound 5a were obtained
at 30 °C. Mass spectra were registered on a COMPACT
MALDI-4 mass spectrometer (Kratos Analytical). UV spectra
were recorded on a Shimadzu UV-1201 spectrophotometer
(J apan) in methanol at pH 7.0. The λmax values were 266-267
nm and ꢀ 9100-9700 for all the synthesized compounds. CD
spectra were recorded on a J -715 spectropolarimeter (J asco,
J apan) in a 1 cm cell. The nucleotide concentration was 0.1-
2
0
capable of cleaving nucleoside 5′-phosphonates. We
also observed hydrolysis of amides 5a and 6a in human
blood serum, which, is likely carried out by phos-
phatases.
0
.2 mM. The results were expressed in the molar circular-
2
dichroic absorption ∆ꢀ ) θ/(32980Cl) cm /mol, where θ was
the measured ellipticity in degrees, C was molar concentration
Modifications of the nucleosides with a phosphono-
formate residue contributed to the anti-HIV efficacy
more noticeably for AZT-containing compounds by af-
fecting to a greater degree level of the activity rather
than the toxicity (Table 3). In the d4T series, most of
the prepared phosphonates were more toxic than d4T.
Therefore, although amides 6a ,b showed higher activity
than the parent nucleoside, the increase in toxicity
lowered their antiviral effect. We believe that an
increase in antiviral activity can be attributed to a
higher intracellular concentration of the derivatives
under study if compared with that of the parent nucleo-
sides.
(mol/L), and l was optical path in centimeters.
Human blood serum was a kind gift of Dr. S. A. Grachev
(Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences). Calf spleen phosphodiesterase
(
17 U/mg) was from Worthington, and crystalline Crotalus
atrox snake venom and Penicillum citrum P1 nuclease (1200
U/mg) were from Sigma.
3
′-Azid o-3′-d eoxyth ym id in e 5′-(eth yl)(eth oxyca r bon yl)
p h osp h on a te 1 was prepared from AZT and triethylphospho-
noformate according to11 (method A) in a yield of 68%. H NMR
1
(CD
.27 (1H, t, J 7.5, H1′), 4.40 (1H, m, H3′), 4.31 (4H, m, 2CH
CH ), 4.20 (2H, m, H5′), 4.07 (1H, m, H4′), 2.25 (2H, m, H2′),
3
CN; δ, ppm; J , Hz): 8.98 (1H, s, NH), 7.43 (1H, m, H6),
6
2
-
3
3
1
1
.88 (3H, s, CH
3
-Thy), 1.57 and 1.52 (6H, 2m, 2CH
2
CH
3
).
P
Although we could not observe a direct correlation
between the stability and antiviral activity of the
synthesized compounds, it is obvious that the analogues
with too high as well as with too low levels of stability
cannot be regarded as promising antivirals. It is likely
that the synthesized phosphonoacetates did not show a
noticeable antiviral effect in cell culture because of the
lack of the enzymes, which could hydrolyze these
structures. To conclude, AZT and d4T modified at 5′-
position with an aminocarbonylphosphonyl fragment
NMR (CD
3
CN; δ, ppm; J , Hz): -4.84 s, -4.37 s. Mass: m/e
+
[M ] 431.1.
Method B. A solution of phosphonate 9 (80 mg, 0.2 mmol)
in pyridine (3 mL), ethanol (0.5 mL) and TPSCl (120 mg, 0.4
mmol) were stirred for 16 h at room temperature and evapo-
rated, and the residue was purified by silica gel column
chromatography in a chloroform-methanol (96:4) system to
give 58 mg (67%) of 1.
2
′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(eth yl)(eth oxy-
ca r bon yl)p h osp h on a te 2 was prepared from d4T similarly
to triester 1 by method A in a yield of 65%. H NMR (CDCl ):
3
1

3
612 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Shirokova et al.
8
6
.50 (1H, s, NH), 7.29 and 7.28 (1H, 2s, H6), 7.01 (1H, m, H1′),
.33 (1H, m, H2′), 5.90 (1H, m, H3′), 5.00 (1H, m, H4′), 4.43
n oca r bon yl)p h osp h on a te 6a . Method C. Triphenylphos-
phine (130 mg, 0.5 mmol) and ethanol (100 µL) were added to
a solution of phosphonate 16 (170 mg, 0.5 mmol) in DMF (2
mL), the solution was cooled to 0 °C, and DEAD (0.5 mmol,
77 µL) solution in THF (1 mL) was added. The reaction
mixture was kept at room temperature for 5 h and evaporated;
the residue was dissolved in water (5 mL) and extracted with
chloroform (3 mL × 3). The aqueous layer was chromato-
graphed on a LiChroprep RP-8 column eluting in a gradient
(2H, m, H5′), 4.31 (4H, m, 2CH
2
CH
3
), 1.89 (3H, s, CH
3
-Thy),
). P NMR (CDCl ):
3.56 s, -3.85 s. Mass: m/e [M ] 388.1.
′-Azid o-3′-d eoxyth ym id in e 5′-(eth yl)(eth oxyca r bon -
3
1
1
-
.38 and 1.33 (6H, 2t, J 7.2, 2CH
2
CH
3
3
+
3
ylm eth yl)p h osp h on a te 3a was prepared from AZT and
triethylphosphonoacetate under conditions of method A in a
yield of 72%. H NMR (CDCl
1
3
): 9.52 and 9.50 (1H, s, NH),
7
.45 and 7.37 (1H, 2s, H6), 6.21 (1H, t, J 6.5, H1′), 4.33 (3H,
m, H3′ + H5′), 4.17 (4H, m, 2CH CH ), 4.01 (1H, m, H4′), 3.01
2H, d, J 21.2, CH P), 2.37 (2H, m, H2′), 1.91 and 1.90 (3H,
s, CH -Thy), 1.34 and 1.26 (6H, 2t, J 7.2, 2CH
CDCl
′-Azid o-3′-d eoxyth ym id in e 5′-(cycloh exyl)(eth oxyca r -
of MeOH in water (0-10%) to give 124 mg (69%) of phospho-
1
nate 6a . H NMR (CD
3
OD): 7.35 (1H, s, H6), 6.96 (1H, m, H1′),
2
3
(
2
(
2
6.37 (1H, m, H2′), 5.94 (1H, m, H3′), 5.02 (1H, m, H4′), 4.37
(2H, m, H5′), 4.19 (2H, m, CH CH ), 1.88 (3H, s, CH -Thy),
). P NMR (CD OD): 0.15 s, -0.12 s.
Mass: m/e [M ] 359.1.
3
1
3
2
CH
): 21.44 s, 20.82 s. Mass: m/e [M ] 445.1.
3
). P NMR
2
3
3
+
31
1.35 (3H, m, CH
2
CH
3
3
3
+
3
bon ylm eth yl)p h osp h on a te 3b was prepared from phospho-
2′,3′-Did eoxy-2′,3′-d id eh ydr oth ym id in e 5′-(eth yl)(m eth -
yla m in oca r bon yl)p h osp h on a te 6b was obtained from 2′,3′-
dideoxy-2′,3′-didehydrothymidine 5′-(methylaminocarbonyl)-
phosphonate 17 and ethanol by method C and was isolated
by chromatography on a silica gel column (2.5 × 30 cm) eluting
nate 11 and cyclohexanol under conditions of method B in a
1
yield of 46%. H NMR (CD
3
CN): 9.41 (1H, s, NH), 7.45 and
7
.38 (1H, 2q, J 1.2, H6), 6.16 and 6.15 (1H, 2t, J 6.8, H1′),
.46 (1H, m, CH (cyclohexyl)) 4.37 (1H, m, H3′), 4.25 (2H, m,
CH ), 4.03 (1H, m, H4′), 3.03 and 3.02
P), 2.38 (2H, m, H2′), 1.90 (2H, m,
cyclohexyl), 1.86 (3H, br.s, CH -Thy), 1.70, 1.50, 1.32 (8H, 3m,
cyclohexyl), 1.24 and 1.23 (3H, 2t, J 7.2, CH
4
H5′), 4.14 (2H, m, CH
2H, 2d, J 21.5, CH
2
3
in a gradient of MeOH in chloroform (0-10%) to give 48% of
1
(
2
phosphonate 6b. H NMR (CD
3
CN): 8.92 (1H, s, NH-Thy), 7.53
(1H, m, CH
6.28 (1H, m, H2′), 5.88 (1H, m, H3′), 4.97 (1H, m, H4′), 4.41
(2H, m, H5′), 4.19 (2H, m, CH CH ), 2.87 and 2.86 (3H, s,
CH N), 1.91 (3H, s, CH -Thy), 1.34 (3H, m, CH
CD CN): -0.27 s, -0.76 s. Mass: m/e [M ] 373.1.
2′,3′-Did eoxy-2′,3′-d id eh yd r ot h ym id in e 5′-(et h yl)[(2-
3
NH), 7.33 and 7.31 (1H, 2s, H6), 7.00 (1H, m, H1′),
3
3
1
2
CH
CN): 20.83 s, 20.52 s. Mass: m/e [M ] 499.2.
′,3′-Dideoxy-2′,3′-dideh ydr oth ym idin e 5′-(eth yl)(eth oxy-
3
). P NMR
+
(
CD
2
3
2
3
3
1
3
3
2 3
CH ). P NMR
+
(
3
ca r bon ylm eth yl)p h osp h on a te 4a was prepared from d4T
and triethylphosphonoacetate by method A in a yield of 67%.
1
H NMR (CDCl
3
): 8.98 (1H, s, NH), 7.34 and 7.27 (1H, 2s,
p h en yleth yl)a m in oca r bon yl]p h osp h on a te 6c was pre-
pared from 2′,3′-dideoxy-2′,3′-didehydrothymidine 5′-[(2-phen-
H6), 7.00 (1H, m, H1′), 6.32 (1H, m, H3′), 5.88 (1H, m, H2′),
4
2
1
2
.99 (1H, m, H4′), 4.49-4.28 (2H, m, H5′), 4.17-4.12 (4H, m,
CH CH ), 2.97-2.90 (2H, m, CH P), 1.90 (3H, s, CH -Thy),
.30 and 1.25 (6H, 2t, J 7.2, 2CH ). P NMR (CDCl ):
ylethyl)aminocarbonyl]phosphonate 18 and ethanol in a yield
1
2
3
2
3
of 51% by method C and 72% by method D. H NMR (CDCl
3
):
3
1
2
CH
1.21 s, 21.03 s. Mass: m/e [M ] 402.1.
′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(cycloh exyl)
eth oxyca r bon ylm eth yl)p h osp h on a te 4b. Phosphonate 10
3
3
2
9.17 (1H, s, NH-Thy), 7.45 (1H, s, CH NH), 7.33 and 7.31 (1H,
2s, H6), 7.45-7.17 (5H, m, Ph), 6.99 (1H, br.s, H1′), 6.27 and
6.17 (1H, 2m, H2′), 5.84 (1H, m, H3′), 4.91 (1H, m, H4′), 4.29
+
2
(
(2H, m, H5′), 4.09 (2H, m, CH
(2H, t, J 7, CH Ph), 1.90 and 1.91 (3H, 2s, CH
m, CH CH ). Mass: m/e [M ] 463.2.
2
CH
3
), 3.62 (2H, m, CH
2
N), 2.85
was treated with cyclohexanol under the conditions described
2
3
-Thy), 1.29 (3H,
in method B to give target 4b in a yield of 39%. 1H NMR
+
2
3
(
CDCl
3
): 8.98 (1H, s, NH), 7.34 and 7.27 (1H, 2s, H6), 7.00
3′-Azid o-3′-d eoxyth ym id in e 5′-(eth yl)(a m in oca r bon yl-
m eth yl)p h osp h on a te 7 was prepared from phosphonate 19
and ethanol by method C in a yield of 77%. H NMR
(1H, m, H1′), 6.32 (1H, m, H3′), 5.88 (1H, m, H2′), 4.99 (1H,
1
m, H4′), 4.49-4.28 (2H, m, H5′), 4.22 (1H, m, CH (cyclohexyl)),
CH P), 1.90
4
.17-4.12 (2H, m, CH
2
3
), 2.97-2.90 (2H, m, CH
2
(CD
H6), 6.68 (1H, s, Ha (NH
6.12 and 6.13 (1H, 2t, J 6.5, H1′), 4.47 and 4.42 (1H, 2m, H3′),
4.31-4.23 (2H, m H5′), 4.15 (2H, m, CH CH ), 4.03 (1H, m,
H4′), 2.94 and 2.93 (2H, 2d, J 21.5, CH P), 2.39 (2H, m, H2′),
1.84 (3H, br.s, CH -Thy), 1.30 (3H, t, J 7, CH
(CD CN): 24.36s, 24.03 s. Mass: m/e [M ] 416.1.
3
CN): 10.03 (1H, s, NH-Thy), 7.48 and 7.44 (1H, 2q, J 1,
(3H, s, CH
3
-Thy), 1.70, 1.58-1.48, 1.33, 1.28 (10H, 4m, 5CH
2
2
)), 6.44 and 6.43 (1H, 2s, Hb (NH )),
2
3
1
(cyclohexyl)), 1.25 (3H, m, CH
2
CH
3
). P NMR (CDCl
3
): 19.91
+
s, 19.74 s. Mass: m/e [M ] 456.16.
2
3
3
′-Azid o-3′-d eoxyth ym id in e 5′-(eth yl)(a m in oca r bon yl)-
2
3
1
p h osp h on a te 5a was prepared from compound 13 and
ethanol by method B in a yield of 72%. H NMR (CD
3
2 3
CH ). P NMR
1
+
3
CN): 9.43
3
(
1H, s, NH-Thy), 7.41 and 7.39 (1H, 2q, J 1, H6), 7.24 (1H,
2′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(eth yl)(a m i-
n oca r bon ylm eth yl)p h osp h on a te 8 was prepared from 2′,3′-
dideoxy-2′,3′-didehydrothymidine 5′-(aminocarbonylmethyl)-
3
2 2
br.s, Ha (NH )), 6.83 (1H, d, J H-P 34.9, Hb (NH )), 6.14 (1H,
t, J 6.5, H1′), 4.41 (1H, m, H3′), 4.34 (2H, m, H5′), 4.21 (2H,
m, 2CH CH ), 4.03 (1H, m, H4′), 2.39 (2H, m, H2′), 1.85 (3H,
m, CH -Thy), 1.33 and 1.32 (3H, 2t, J 7, 2CH
CD CN): -0.08 s, -0.35 s. Mass: m/e [M ] 402.1.
′-Azido-3′-deoxyth ym idin e 5′-(eth yl)(m eth ylam in ocar -
bon yl)p h osp h on a te 5b was prepared from compound 14 and
2
3
phosphonate 20 and ethanol by method C in a yield of 67%.
3
1
1
3
2
CH
3
). P NMR
3
H NMR (CD CN): 10.02 (1H, s, NH-Thy), 8.78 (1H, s, NH),
+
(
3
7.32 and 7.29 (1H, 2s, H6), 6.89 (1H, m, H1′), 6.37 (1H, m,
H3′), 5.92 (1H, m, H2′), 4.98 (1H, m, H4′), 4.28-4.18 (2H, m,
3
H5′), 4.17-4.02 (2H, m, CH
1.85 and 1.84 (3H, 2s, CH -Thy), 1.25 (6H, m, CH
NMR (CD CN): 25.36 s, 25.16 s. Mass: m/e [M ] 373.1.
2
CH
3
2
), 2.85-2.79 (2H, m, CH P),
1
31
ethanol by method B in a yield of 64%. H NMR (CD
3
CN): 9.47
NH), 7.43 (1H, s, H6), 6.22
1H, t, J 6.8, H1′), 4.40 (3H, m, H3′ + H5′), 4.22 (2H, m, CH
3
2
CH
3
).
P
+
(
(
1H, s, NH-Thy), 7.72 (1H, m, CH
3
3
2
-
3′-Azid o-3′-d eoxyth ym id in e 5′-(eth oxyca r bon yl)p h os-
p h on a te 9. To a solution of AZT (133 mg, 0.5 mmol) and
pyridinium salt of ethoxycarbonylphosphonic acid (151 mg,
0.65 mmol) in pyridine (4 mL) was added DCC (436 mg, 2
mmol), and the suspension was stirred for 18 h at room
temperature. The mixture was diluted with water (10 mL),
the precipitate was filtered, and the filtrate was purified on a
CH
3
), 4.01 (1H, m, H4′), 2.91 and 2.89 (3H, 2s, CH
3
N), 2.39
(2H, m, H2′), 1.94 and 1.93 (3H, 2s, CH
3
-Thy), 1.37 (3H, m,
CH ). P NMR (CD CN): -0.10 s, -0.54 s. Mass: m/e [M ]
3 3
3
1
+
CH
4
2
16.1.
3
′-Azid o-3′-d eoxyth ym id in e 5′-(eth yl)[(2-p h en yleth yl)-
a m in oca r bon yl]p h osp h on a te 5c was prepared from com-
pound 15 and ethanol by method B in a yield of 70%. H NMR
1
DEAE-Toyopearl column in a linear gradient of NH
4 3
HCO (0-
(
CD
and 7.40 (1H, 2s, H6), 7.25 (5H, m, Ph), 6.15 (1H, t, J 6.8,
H1′), 4.33 (1H, m, H3′), 4.27 (2H, m, H5′), 4.12 (2H, m, CH
3 2
CN): 9.43 (1H, s, NH-Thy), 7.62 (1H, m, CH NH), 7.41
0.15 M). Compound 9 was obtained in a yield of 169 mg (81%).
1
H NMR (D
(1H, m, H3′), 4.10 (2H, m, CH
2.38 (2H, m, H2′), 1.78 (3H, s, CH
3
2
O): 7.58 (1H, s, H6), 6.15 (1H, t, J 7.5, H1′), 4.40
CH ), 4.07 (3H, m, H4′ + H5′),
-Thy), 1.11 (3H, t, J 7.1,
2
-
2
3
CH
CH
3
), 4.00 (1H, m, H4′), 3.53 (2H, s, CH
2
NH), 2.83 (2H, t, J 7,
-Thy),
CN): 0.04 s, -0.40 s.
3
1
2
Ph), 2.36 (2H, m, H2′), 1.86 and 1.84 (3H, 2s, CH
3
2 3 2
CH CH ). P NMR (D O): -4.93 s.
3
1
1
.28 (3H, m, CH
2
CH
Mass: m/e [M ] 506.2.
′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(Eth yl)(a m i-
3
). P NMR (CD
3
2′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-eth oxyca r -
bon ylp h osp h on a te 10 was obtained from d4T and pyri-
dinium salt of ethoxycarbonylphosphonic acid under the
+
2

Anti-HIV Pronucleosides
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3613
1
conditions described for 9 in a yield of 79%. H NMR (D
2
O):
.54 (1H, q, J 1, H6), 6.78 (1H, m, H1′), 6.31 (1H, m, H2′),
.79 (1H, m, H3′), 4.91 (1H, m, H4′), 3.98 (4H, m, H5′ + CH
column in a linear gradient of NH
was 86%. H NMR (D
2
4
HCO
O): 7.43 (1H, s, H6), 7.40-7.17 (5H, m,
Ph), 6.89 (1H, m, H1′), 6.25 (1H, m, H2′), 5.86 (1H, m, H3′),
4.95 (1H, m, H4′), 4.10 (2H, m, H5′), 3.60 (2H, m, CH N), 2.61
O): -1.47
3
(0-0.2 M). The yield
1
7
5
2
-
). 1P
3
CH
NMR (D
′-Azid o-3′-d eoxyth ym id in e 5′-(eth oxyca r bon ylm eth -
3
), 1.75 (3H, d, CH
3
-Thy), 1.04 (3H, t, J 7.2, CH
2
CH
3
2
3
1
2
O): -5.16 s.
2 3 2
(2H, t, J 7, CH Ph), 1.86 (3H, s, CH ). P NMR (D
s.
3
yl)p h osp h on a te 11 was prepared from AZT, pyridinium salt
3′-Azid o-3′-d eoxyt h ym id in e 5′-(a m in oca r b on ylm et h -
yl)p h osp h on a te 19. A solution of phosphonate 11 (85 mg,
0.2 mmol) in 25% aqueous ammonia (3 mL) was kept for 18 h
at 20 °C and evaporated. The residue was dissolved in water
and purified on a DEAE-Toyopearl column in a linear gradient
of ethoxycarbonylmethylphosphonic acid, and DCC similarly
1
to 9 in a yield of 69%. H NMR (D
2
O): 7.77 (1H, s, H6), 6.32
(
1H, t, J 6.6, H1′), 4.55 (1H, m, H3′), 4.27-4.19 (5H, m, H4′ +
H5′ + CH CH ), 2.96 (2H, d, J 20.6, CH P), 2.57 (2H, m, H2′),
.99 (3H, br.s, CH -Thy), 1.31 (3H, t, J 7.2, CH
2
3
2
3
1
1
3
2
CH
3
). P NMR
of NH
4
HCO
19. H NMR (D
.35 (1H, m, H3′), 4.03-4.00 (3H, m, H4′ + H5′), 2.63 (2H, d,
3
(0-0.1 M) to give 54 mg (68%) of phosphonate
2
1
(D
2
O): 15.09 s.
O): 7.56 (1H, s, H6), 6.10 (1H, t, J 6.6, H1′),
4
2
′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(eth oxyca r -
3
1
J 20.6, CH
O): 16.26 s.
2′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(Am in oca r -
bon ylm eth yl)p h osp h on a te 20. Treatment of 2′,3′-dideoxy-
′,3′-didehydrothymidine 5′-(ethoxycarbonylmethyl)phospho-
2 3
P), 2.34 (2H, m, H2′), 1.76 (3H, s, CH ). P NMR
bon ylm eth yl)p h osp h on a te 12 was obtained from d4T and
(D
2
pyridinium salt of ethoxycarbonylmethylphosphonic acid under
the conditions described for 9 in a yield of 78%. 1H NMR
(D
2
O): 7.45 (1H, q, J 1, H6), 6.78 (1H, m, H1′), 6.31 (1H, m,
H2′), 5.79 (1H, m, H3′), 4.91 (1H, m, H4′), 3.93 (4H, m, H5′ +
CH CH ), 2.63 (2H, d, J 20.4, CH P), 1.72 (3H, d, CH -Thy),
.04 (3H, t, J 7.2, CH ). P NMR (D O): 14.86 s.
′-Azid o-3′-d eoxyth ym id in e 5′-(a m in oca r bon yl)p h os-
2
nate 12 with 25% ammonia yielded 94% of 2′,3′-dideoxy-2′,3′-
2
3
2
3
3
1
didehydrothymidine 5′-(aminocarbonylmethyl)phosphonate 20.
1
2
CH
3
2
1
2
H NMR (D O): 7.45 (1H, s, H6), 6.78 (1H, m, H1′), 6.31 (1H,
3
m, H2′), 5.79 (1H, m, H3′), 4.91 (1H, m, H4′), 3.93 (2H, m,
p h on a te 13. A solution of phosphonate 9 (80 mg, 0.2 mmol)
in 25% aqueous ammonia (3 mL) was kept for 18 h at 20 °C
and evaporated. The residue was dissolved in water and
purified on a DEAE-Toyopearl column in a linear gradient of
3
1
H5′), 2.63 (2H, d, J 20.4, CH
NMR (D O): 16.86 s.
2
P), 1.72 (3H, br.s, CH
3
-Thy).
P
2
Sta bility Stu d ies. Hydrolysis was carried out in the 0.05
M phosphate buffer at pH 7.2 and 20 °C at the concentrations
of the compounds under study of 0.5 mM. The aliquots (10 µL)
were taken out after certain intervals and frozen in liquid
nitrogen, and the products were analyzed by HPLC.
NH
4
HCO
H NMR (DMSO-d
NH
H4′ + H5′), 2.30 (2H, m, H2′), 1.81 (3H, br.s, CH
NMR (DMSO-d ): -1.56 s.
′-Azid o-3′-d eoxyt h ym id in e 5′-(m et h yla m in oca r bon -
3
(0-0.1 M) to give 70 mg (94%) of phosphonate 13.
1
6
): 7.82 (1H, m, H6), 7.17 and 7.13 (2H, 2s,
2
), 6.12 (1H, t, J 6.9, H1′), 4.50 (1H, m, H3′), 3.95 (3H, m,
-Thy). 31
3
P
The chemical stability of amide 5a was also studied in 0.05
M sodium phosphate buffer at 37 °C and pH in the range of
6
3
5
.5-7.8 (the concentration of amide 5a was 0.45 mM). The
yl)p h osp h on a te 14. A solution of phosphonate 9 (80 mg, 0.2
mmol) in 25% aqueous methylamine (3 mL) was kept for 1 h
at 20 °C and evaporated. The residue was dissolved in water
and purified on a DEAE-Toyopearl column in a linear gradient
reaction mixture was treated and analyzed by HPLC under
the conditions described above.
The enzymatic hydrolysis was studied in the mixture (200
µL) containing 0.1 M Tris-HCl buffer (pH 7.5), 5 mM MgCl ,
2
of NH
4
HCO
4. H NMR (D
.32 (1H, m, H3′), 4.01 (3H, m, H4′ + H5′), 2.61 (3H, s, CH
3
(0-0.2 M) to give 71 mg (91%) of phosphonate
O): 7.52 (1H, s, H6), 6.06 (1H, t, J 6.7, H1′),
N),
O): -1.40
and 0.45 mM amide 5a , incubated at 37 °C in the presence of
either snake venom phosphodiesterase (0.1 mg/mL) or Crotalus
atrox snake venom (1 mg/mL). The reaction mixture was
treated and analyzed by HPLC under the conditions described
above.
The reaction mixture (200 µL) containing 0.1 M acetate
buffer (pH 6.0), 0.45 mM amide 5a , and calf spleen phosphodi-
esterase (0.5 mg/mL) was incubated at 37 °C. The reaction
mixture was treated and analyzed by HPLC as described
above.
1
1
4
2
2
3
3
1
3 2
.32 (2H, m, H2′), 1.72 (3H, s, CH -Thy). P NMR (D
s.
3
′-Azid o-3′-d eoxyt h ym id in e 5′-[(2-P h en ylet h yl)a m i-
n oca r bon yl]p h osp h on a t e 15. A solution of 9 (80 mg, 0.2
mmol) in 2-phenylethylamine (1 mL) was stirred for 4 h at 20
°
C and evaporated. The residue was dissolved in water and
purified on a DEAE-Toyopearl column in a linear gradient of
NH HCO (0-0.15 M) to give 82 mg (86%) of phosphonate 15.
H NMR (D O): 7.42 (1H, s, H6), 7.17-7.05 (5H, m, Ph), 6.02
1H, t, J 6.5, H1′), 4.17 (1H, m, H3′), 3.90 (1H, m, H4′), 3.81
2H, m, H5′), 3.39 (2H, m, CH N), 2.69 (2H, t, J 6.5, CH Ph),
.24 (2H, m, H2′), 1.68 (3H, s, CH O): -1.57 s.
′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(a m in oca r -
4
3
The reaction mixture (200 µL) containing 0.02 M Tris-HCl
buffer (pH 7.2), 0.45 mM amide 5a , and P1 nuclease (0.1 mg/
mL) was incubated at 37 °C. The reaction mixture was treated
and analyzed by HPLC as described above.
1
2
(
(
2
2
2
3
1
3 2
). P NMR (D
An tivir a l Activity. MT-4 cells were infected with HIV-1
strain (GKV-4046). The multiplicity of infection was 0.2-0.5
units per cell. MT-4 cells were maintained in RPMI-1640
medium supplemented with 10% fetal bovine serum, 300 µg/
mL L-glutamine, 80 µg/mL gentamycin, and 30 µg/mL linco-
2
bon yl)p h osp h on a te 16. Treatment of 2′,3′-dideoxy-2′,3′-
didehydrothymidine 5′-(ethoxycarbonyl)phosphonate 10 with
2
9
6
5% ammonia analogously to the preparation of 13 yielded
1
6% of the title phosphonate. H NMR (D
.88 (1H, m, H1′), 6.41 (1H, m, H2′), 5.91 (1H, m, H3′), 5.04
-Thy). 31
2
O): 7.51 (1H, s, H6),
2
mycin at 37 °C in 5% CO atmosphere. For infecting, the cells
6
at the concentration 2 × 10 cell/mL and viability exceeding
(1H, m, H4′), 4.09 (2H, m, H5′), 1.81 (3H, br.s, CH
3
P
9
0% were used.
NMR (D O): -1.62 s.
2
Cytotoxicity. MT-4 cells were cultured in the presence of
various doses of the tested compounds (0.001-200 µg/mL,
three replicates for each dose), on a 96-well cultural plate for
2
′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-(Meth yla m i-
n oca r bon yl)p h osp h on a te 17. Treatment of 2′,3′-dideoxy-
′,3′-didehydrothymidine 5′-(ethoxycarbonyl)phosphonate 10
with 25% aqueous MeNH yielded 92% of 2′,3′-dideoxy-2′,3′-
didehydrothymidine 5′-(methylaminocarbonyl)phosphonate 17,
2
3
days. The concentration and viability of MT-4 cells were
2
measured by the trypan blue-dye exclusion colorimetric assay,
and the CD50 for each compound was calculated.
1
which was further used without purification. H NMR (D
.43 (1H, s, H6), 6.85 (1H, m, H1′), 6.35 (1H, m, H2′), 5.86
1H, m, H3′), 4.97 (1H, m, H4′), 4.03 (2H, m, H5′), 2.61 (3H, s,
2
O):
An ti-HIV Assa y. The infected cells were cultured in the
presence of various doses of the tested compounds (0.001-100
µg/mL, three replicates for each dose) on a 96-well cultural
plate for 3 days. Anti-HIV activity of the tested compounds
was assessed by the measurement of p24 antigen amount
using immunoassay.23 The cell concentration and viability were
estimated using the colorimetric assay as described above.
7
(
3
1
CH
3
N), 1.76 (3H, s, CH
3 2
-Thy). P NMR (D O): -1.53 s.
2
′,3′-Did eoxy-2′,3′-d id eh yd r oth ym id in e 5′-[(2-P h en yl-
eth yl)a m in oca r bon yl]p h osp h on a te 18. Treatment of 2′,3′-
dideoxy-2′,3′-didehydrothymidine 5′-(ethoxycarbonyl)phospho-
nate 10 with 2-phenylethylamine yielded crude 2′,3′-dideoxy-
2
′,3′-didehydrothymidine 5′-[(2-phenylethyl)aminocarbonyl]-
Ack n ow led gm en t. The work was supported by the
phosphonate 18, which was purified on a DEAE-Toyopearl
Russian Foundation for Basic Research, projects 02-04-

3
614 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Shirokova et al.
4
9009 and 03-04-06712; Russian State AIDS program,
(12) Lambert, R. W.; Martin, J . A.; Thomas, G. J .; Duncan, I. B.; Hall,
M. J .; Heimer, E. P. Synthesis and antiviral activity of phospho-
noacetic and phosphonoformic acid esters of 5-bromo-2′-deox-
yuridine and related pyrimidine nucleosides and acyclonucleo-
sides. J . Med. Chem. 1989, 32, 367-374.
13) J asko, M.; Shipitsin, A.; Shirokova, E.; Kraevsky, A.; Polsky, B.;
Baron, P.; MacLow, C.; Ostrander, M.; O’Hara, B. 5′-Phospho-
nates of ribonucleosides and 2′-deoxyribonucleosides: synthesis
and antiviral activity. Nucleosides Nucleotides 1993, 12, 879-
893.
(14) Karpenko, I. L.; J asko, M. V.; Andronova, V. L.; Ivanov, A. V.;
Kukhanova, M. K.; Galegov, G. A.; Skoblov, Y. S. Synthesis and
antiherpetic activity of acyclovir phosphonates. Nucleosides,
Nucleotides Nucleic Acids 2003, 22, 319-328.
(15) Campbell, D. A. The synthesis of phosphonate esters, an
extension of the Mitsunobu reaction. J . Org. Chem. 1992, 57,
6331-6335.
(16) Miles, D. W.; Robins, M. J .; Robins, R. K.; Winkley, M. W.;
Eyring, H. Circular dichroism of nucleoside derivatives. V.
Cytosine derivatives. J . Am. Chem. Soc. 1969, 91, 831-838.
(17) Rozowsky, A.; Fu, H.; Pai, N.; Mellors, J .; Richman, D. D.;
Hostetler, K. Y. Synthesis and in vitro activity of long-chain 5′-
O-((alkoxycarbonyl)phosphinyl)-3′-azido-3′-deoxythymidines
against wild-type and AZT- and Foscarnet-resistant strains of
HIV-1. J . Med. Chem. 1997, 40, 2482-2490.
(18) Meier, C.; Aubertin, A. M.; de Monte, M.; Faraj, A.; Sommadossi,
J . P.; Perigaud, C.; Imbach, J . L.; Gosselin, G. Synthesis and
antiviral evaluation of SATE-foscarnet prodrugs and new fos-
carnet-AZT conjugates. Antivir. Chem. Chemother. 1998, 9, 41-
52.
(19) Noren, J . O.; Helgstrand, E.; J ohansson, N. G.; Misiorny, A.;
Stening G. Synthesis of esters of phosphonoformic acid and their
antiherpes activity. J . Med. Chem. 1983, 26, 264-270.
(20) Kukhanova, M. K.; Tarussova, N. B.; J asko, M. V.; Arzumanov,
A. A.; Gudima, S. O.; Kraevsky, A. A.; Chidgevadze, Z. G.;
Beabealashvilli, R. Sh. Hydrolysis of 5′-phosphonates by phos-
hatases of different origin and human or calf blood sera.
Molekulyarnaya Biol. (Russian) 1992, 26, 1148-1159.
(21) Plotto, M.; Saudek, V.; Sklenar V. Gradient tailored excitation
for single-quantum NMR spectroscopy of aqueous solutions. J .
Biomol. NMR 1992, 2, 661-665.
project 31; and the program of Presidium of Russian
Academy of Sciences (Physicochemical Biology).
(
Refer en ces
(
(
(
1) De Clercq, E. New development in anti-HIV chemotherapy.
Biochim. Biophys. Acta 2002, 1587, 258-275.
2) Zemlicka, J . Lipophilic phosphoramidates as antiviral pronucle-
otides. Biochim. Biophys. Acta 2002, 1587, 276-286.
3) Perigaud C.; Gosselin G.; Imbach J .-L. Minireview: from the
pronucleotide concept to the SATE phosphate protecting groups.
Curr. Top. Med. Chem. 1997, 2, 15-29.
(
4) Siddiqui, A. Q.; McGuigan, C.; Ballatore, C.; Zuccotto, F.; Gilbert,
I. H.; De Clercq, E.; Balzarini, J . Design and synthesis of
lipophilic phosphoramidate d4T-MP prodrugs expressing high
potency against HIV in cell culture: structural determinants
for in vitro activity and QSAR. J . Med. Chem. 1999, 42, 4122-
4
128.
(
5) Wagner, C. R.; Iyer V. V.; McIntee, E. J . Pronucleotides: toward
the in vivo delivery of antiviral and anticancer nucleotides. Med.
Res. Rev. 2000, 20, 417-451
(
6) Mavromoustakos, T.; Calogeropoulou, T.; Koufaki, M.; Kolo-
couris, A.; Daliani, I.; Demetzos, C.; Meng, Z.; Makriyannis, A.;
Balzarini, J .; De Clercq, E. Ether phospholipid-AZT conjugates
possessing anti-HIV and antitumor cell activity. Synthesis,
conformational analysis, and study of their thermal effects on
membrane bilayers. J . Med. Chem. 2001, 44, 1702-1709.
7) Balzarini, J .; Aquaro, S.; Knispel, T.; Rampazzo, C.; Bianchi, V.;
Perno, C. F.; De Clercq, E.; Meier, C. Cyclosaligenyl-2′,3′-
didehydro-2′,3′-dideoxythymidine monophosphate: efficient in-
tracellular delivery of d4TMP. Mol. Pharmacol. 2000, 58, 928-
(
(
(
3
5.
8) Cardona, V. M.; Ayi, A. I.; Aubertin, A. M.; Guedj, R. Synthesis
and anti-HIV activity of some novel aryl phosphate and H-
phosphonate derivatives of 3′-azido-2′,3′-dideoxythymidine and
2
1
′,3′-didehydro-2′,3′-dideoxythymidine. Antiviral Res. 1999, 42,
89-196.
9) Machado J .; Salomon H.; Oliveira M.; Tsoukas C.; Krayevsky
A. A.; Wainberg M. A. Antiviral activity and resistance profile
of phosphazidsa novel prodrug of AZT. Nucleosides Nucleotides
(22) Cavanagh, J .; Fairbrother, W. J .; Palmer, A. G.; Skelton, N. J .
Protein NMR spectroscopy: principles and practice; Academic
Press: San Diego, 1996.
(23) Svinarchik, F. P.; Konevetz, D. A.; Plyasunova, O. A.; Pokrovsky,
A. G.; Vlassov, V. V. Inhibition of HIV proliferation by antisense
oligonucleotide conjugated to lipophilic groups. Biochimie 1993,
75, 49-54.
1
999, 18, p 901-6.
(
10) Parang, K.; Wiebe, L. I.; Knaus, E. E. Novel approaches for
designing 5′-O-ester prodrugs of 3′-azido-2′,3′-dideoxythymidine
(AZT). Curr. Med. Chem. 2000, 7, 995-1039.
(
11) Rozowsky, A.; Saha, J .; Fazely, F.; Koch, J .; Ruprecht, R. M.
Inhibition of human immunodeficiency virus type 1 replication
by phosphonoformate esters of 3′-azido-3′-deoxythymidine. Bio-
chem. Biophys. Res. Commun. 1990, 172, 288-294.
J M0310176