New phosphonoformic acid derivatives of 3′-azido-3′- deoxythymidine

Article abstract of DOI:10.1023/B:RUBI.0000030131.37092.0a

New 5′-alkyl ethoxy- and aminocarbonylphosphonates of 3′-azido-3′-deoxythymidine (AZT) were synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis were studied. The AZT 5′-aminocarbonylphosphonates

Full text of DOI:10.1023/B:RUBI.0000030131.37092.0a

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 3, 2004, pp. 242–249. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 3, 2004, pp. 273–280.
Original Russian Text Copyright © 2004 by Shirokova, Jasko, Khandazhinskaya, Ivanov, Yanvarev, Skoblov, Pronyaeva, Fedyuk, Pokrovskii, Kukhanova.
New Phosphonoformic Acid Derivatives
of 3'-Azido-3'-Deoxythymidine
1
E. A. Shirokova*, M. V. Jasko*, A. L. Khandazhinskaya*, A. V. Ivanov*,
D. V. Yanvarev*, Yu. S. Skoblov*, T. R. Pronyaeva**, N. V. Fedyuk**,
A. G. Pokrovskii**, and M. K. Kukhanova*
*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
**Vector State Research Center of Virology and Biotechnology, Kol’tsovo, Novosibirsk oblast, 633159 Russia
Received February 3, 2003; in final form, April 18, 2003
Abstract—New 5'-alkyl ethoxy- and aminocarbonylphosphonates of 3'-azido-3'-deoxythymidine (AZT) were
synthesized, and their antiviral properties in HIV-1-infected cell cultures and stability to chemical hydrolysis
were studied. TheAZT 5'-aminocarbonylphosphonates were shown to be significantly more stable in phosphate
buffer (pH 7.2) than the corresponding ethoxycarbonylphosphonates. The therapeutic (selectivity) index of
some of the compounds exceeded that of the parent AZT due to their higher antiviral activity.
Key words: 3'-azido-3'-deoxythymidine, human immunodeficiency virus, nucleoside phosphonates
1
INTRODUCTION
amidodiesters (III) as well as the results of testing their
antiviral activities on cell cultures infected with HIV.
Understanding of the nature of AIDS and the dis-
covery of targets for the antiviral agents allowed the
design of a number of medicines for theAIDS therapy.
The most widely used nucleoside-based anti-HIV drug
is AZT.
2
RESULTS AND DISCUSSION
Triester (IIa) has been previously synthesized by
the reaction of AZT with ethyl ethoxycarbonylchloro-
phosphinate [3]. Although the yield of the product was
60–70%, this method did not allow the variation of sub-
stituents at phosphinate residue.
A general scheme of synthesis of the target triesters
(II) and amidodiesters (III) involved the preparation of
the corresponding charged derivatives (IV) and (V) fol-
lowed by their esterification (scheme).
It was recently shown that some phosphonates of
modified nucleosides display high antiviral activities
and low toxicities. 3'-Azido-3'-deoxythymidine 5'-H-
phosphonate (Nicavir) used nowadays for the treatment
of HIV-infected patients can be regarded as a good
example of application of a nucleoside phosphonate
analogue in medicine [1, 2].
Previously, we had synthesized compounds (IV)
from the corresponding nucleosides and ethoxycarbon-
ylphosphonic acid using N,N'-dicyclohexylcarbodiim-
ide as a coupling reagent [6]. The yields of products did
not exceed 70%. In this work, phosphonoformate (IV)
was obtained by the AZT coupling with ethoxycarbon-
ylphosphonic acid in the presence of pivaloyl chloride
in 85% yield. The interaction of the resulting phospho-
nate (IV) with a number of primary amines led to high
yields of the corresponding amides (V). The reaction
AZT phosphonoformates of type (I) is one of the
groups of compounds directed toward the inhibition of
HIV replication. Compounds (Ia) were synthesized in
expectation that the biological activity of both their
moieties could give rise to a synergetic effect. However,
this effect was not found, although the compounds dis-
played a good activity toward the HIV-infected AZT-
resistant cell cultures [3, 4]. Triesters (Ib) with a signif-
icant lipophilicity demonstrated pronounced anti-HIV
properties; however they were insufficiently stable dur-
ing isolation [5].
We describe in this work the synthesis and stability
of AZT-containing triesters (II) and the corresponding
O
O
1
Corresponding author; phone: +7 (095) 135-6065; fax: +7 (095)
P O AZT
OR'
135-1405; e-mail: shirokova-elena@list.ru
RO
2
Abbreviations: AZT, 3'-azido-3'-deoxythymidine; HIV, human
immunodeficiency virus; CD , a dose causing 50% cell death;
50
ID and ID , doses inhibiting the virus replication by 50 and
50
90
(Ia), R = Me, Et, R' = H
90%, respectively; SI, the selectivity index CD /ID ; and
50
50
TPSCl, 2,4,6-triisopropylbenzenesulfonyl chloride.
(Ib), R = C8H17, (CH3)3CC(O)S(CH2)2–, R' = AZT
1068-1620/04/3003-0242 © 2004 MAIK “Nauka /Interperiodica”

NEW PHOSPHONOFORMIC ACID DERIVATIVES OF 3'-AZIDO-3'-DEOXYTHYMIDINE
243
O
O
O
O
P O
OR
P O
OR
Thy
Thy
EtO
R'
O
O
N3
N3
(IIa), R = Et
(IIIa), R = Et, R' = NH2
(IIb), R = PhCH2CH2
(IIc), R = Pri
(IIIb), R = Me, R' = NH2
(IIIc), R = Pr, R' = NH2
(IIId), R = Pri, R' = NH2
(IId), R =
(IIe), R =
(IIIe), R = (CH3)3CCH2, R' = NH2
(IIIf), R = Et, R' = MeNH
(IIIg), R = Et, R' = PhCH2CH2NH
N
(IIIh), R = Et, R' =
O
with secondary amines proceeded less uniquely. For ence of TPSCl (method A) or under the Mitsunobu
example, the major product isolated in the reaction with
diethylamine was AZT 5'-H-phosphonate, whereas the
reflux of AZT with morpholine led to the target amide
(Vd) in a high yield (77%).
reaction conditions (method B). The drawback of the
first method was the necessity of using a large excess of
coupling agent (3 equiv) because of the side reaction of
formation of the corresponding 2,4,6-triisopropylben-
zenesulfonate of the alcohol used. The Mitsunobu reac-
Phosphonates (IV) and (V) were esterified by the
coupling with the corresponding alcohols in the pres- tion proceeded under neutral conditions, which made it
O
O
O
O
HO
P O
OH
P O
OH
Thy
Thy
Thy
EtO
R'
O
O
O
R'H
N3
N3
N3
(IV)
(Va), R' = NH2
(Vb), R' = MeNH
ROH
O
(Vc), R' = PhCH2CH2NH
O
P O
OR
Thy
N
EtO
O
(Vd), R' =
O
ROH
N3
(IIa)–(IIe)
O
O
P O
OR
Thy
R'
O
N3
(IIIa)–(IIIh)
Scheme.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

244
SHIROKOVA et al.
–0.928 ppm
–1.241 ppm
–0.819 ppm –1.037 ppm
35 Hz
(a)
(b)
–0.8
–1.0
–1.2
–1.4 –0.6
ppm
–0.8
–1.0
–1.2
–1.4
ppm
31
Fig.1. P NMR spectra of amide (IIIa) enriched with one of the diastereomers registered (a) with and (b) without phosphorus–
proton decoupling.
attractive for the synthesis of pH-sensitive compounds. thymine protons, which are as a rule represented as two
However, the product yields from secondary alcohols sets of resonances (see the Experimental section).
1
are considerably lower than those in the case of primary
alcohols [7].
Note that, in the H NMR spectral pattern of (III),
the amide resonances of the NH2CO group are
observed as two groups: a singlet at 7.24 ppm and a
Structures of the resulting compounds were con-
doublet at 6.83 ppm (3JP–H = 35 Hz). The silica gel chro-
1
firmed by H and 31P NMR spectroscopy and mass-
spectrometry (see the Experimental section); their UV matography allowed us to obtain the fraction enriched
with one of the diastereomers (more than 90%). Its 31P
NMR spectrum confirmed the hypothesis on the inter-
action of phosphorus atom with one of the amide pro-
tons with a coupling constant J of 35 Hz (Fig. 1).
spectra agreed with those for thymidine derivatives
(data not given).
A new chiral center (the phosphorus) in compounds
(II) and (III) enables the formation of two diastere-
omers. Actually, two singlet resonances at a ratio of
approximately 1 : 1 were observed for phosphonates
(II) and (III) in the 31P NMR spectra registered with the
phosphorus–proton decoupling. The presence of two
diastereomers is most clearly seen for H6 and 5-ëç3
Stability of the resulting compounds was studied in
phosphate buffer at pH 7.2 and 20°ë (Table 1). The
component composition of the reaction mixture was
analyzed by HPLC on a reversed-phase sorbent. Two
products were found in the case of triesters (II): AZT
and the corresponding ester of AZT H-phosphonate;
authentic samples were synthesized according to the
procedure described in [8]. Amount of the phosphonate
did not exceed 30% from the total amount of products.
Amides (III) were mainly hydrolyzed to the nucleo-
side; the corresponding charged amides (V) were iso-
lated as minor products.
Table 1. The stability of (II) and (III) in phosphate buffer
(pH 7.2) at 37°C. Data were averaged according to three ex-
periments; the experimental error did not exceed 20%
*
τ
1/2 , min
Compound
We used (IIIa) as a model for a thorough study of
the stability of the resulting compounds under varied
conditions. Its transformations were analyzed at 37°ë
and pH 7.2. Two hydrolysis products were found, the
rate of formation of one of them being considerably
higher (Fig. 2). AZT prevailed, and AZT aminocarbon-
ylphosphonate (Va) was a minor product. Authentic
samples of AZT and (Va) were used for the identifica-
tion of hydrolysis products.
(IIa)
10
40
(IIc)
(IIIa)
(IIIc)
(IIId)
(IIIf)
(IIIg)
150
205
390
360
We also studied the chemical stability of (IIIa) in
0.05 M phosphate buffer within the pH range of 5.5–7.8
at 37°ë (Fig. 3). Its half-life at pH 5.5 was about 16 h
ꢀ600
* The time at which 50% of compound was hydrolyzed.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

NEW PHOSPHONOFORMIC ACID DERIVATIVES OF 3'-AZIDO-3'-DEOXYTHYMIDINE
245
and decreased to 34 min at pH 7.8. Within pH 7.2–7.8,
the hydrolysis rate stopped to grow. The half-life of
(IIIa) was approximately 50 min at pH 7.2.
The antiviral activity was examined in MT-4 cells
infected with HIV-1, strain GKV-4046 (Table 2). The
ability of the compounds to inhibit the virus replication
was determined by measuring concentration of the viral
p24 antigen. The compounds were added into the cul-
ture after the virus absorption (see the Experimental
section).
One of the tasks in the design of new antiviral drugs
is currently an increase in the efficiency of the prepara-
tions used. This can be achieved by the proper modifi-
cations of active molecules, which allow an improve-
ment in bioavailability, efficiency of cell penetration,
accelerated transformation into the active form, a
changed catabolism, etc. We believed reasonable to
synthesize AZT phosphonoformates (II) and (III) con-
taining various substituents both at phosphorus atom
and at the carbonyl group and to study their biochemi-
cal and anti-HIV properties.
Note that the compounds bearing the residues of pri-
mary alcohols at the phosphorus atom (IIa) and (IIb)
demonstrated increased antiviral activities; the pres-
ence of residues of secondary alcohols [in (IIc) and
(IId)] increased the toxicities in comparison with the
starting nucleoside; and the introduction of a tertiary
alcohol residue [triester (IIe)] caused a significant
decrease in the anti-HIV activity.
%
100
80
60
40
20
AZT
(IIIa)
(Va)
0
10
20
30
40
5
0
60
min
Fig. 2. Hydrolysis curve of phosphonate (IIIa) at 37°C and
pH 7.2.
t
1/2, h
16
12
8
4
0
All in all, an increase in the anti-HIV activity was
more noticeable for AZT aminocarbonylphosphonates
(III) than for triesters (II) (Table 2).
5.5
6.0
6.5
7.0
7.5
8.0
pH
The hydrolysis of compounds (II) and (III) pro-
ceeded differently. In the case of amidodiesters (III),
AZT was the major product and resulted from the pre-
ferred cleavage of one of its phosphodiester bonds. Tri-
esters (II) were hydrolyzed in a more complicated man-
ner. Along with AZT, P-alkyl esters of AZT 5'-H-phos-
phonate were formed, probably, due to the successive
cleavage of the ethyl residue at the carbonyl group of
triesters (II) and the decarboxylation of the resulting
ethyl 5'-carboxyphosphonate AZT similarly to that
described in [9]. Aminocarbonylphosphonates (III)
turned out to be noticeably more stable in the phosphate
buffer than triesters (II).
Most probably, the phosphonates we synthesized
function in the cell cultures only as AZT depot forms.
Their decreased toxicities and increased antiviral activ-
ities could result from a better bioavailability of these
derivatives. A higher stability can be one of the factors
contributing to an increase in the therapeutic (selectiv-
ity) index of the amide analogues in comparison with
the triesters.
Fig. 3. The dependence of the hydrolysis time of 50% of
amide (IIIa) on pH.
were from Fluka (Switzerland); TPSCl, pyridine, and
DMF from Aldrich (United States); and AZT was a
kind gift of AZT ZAO (Russia).
Adsorption column chromatography was carried out
on Kieselgel 60 (40–60 µm, direct phase) and LiChro-
prep RP-8 and RP-18 (25–40 µm, reversed phase)
(Merck, Germany). DEAE-Toyopearl (HCO–3 ) (Toyo-
soda, Japan) was used for ion-exchange column chro-
matography. HPLC was carried out on a Gilson chro-
matograph supplied with models 307 and 311 pumps
(France) and an LKB 2220 integrator.
NMR spectra were registered on an AMX III-400
spectrometer (Bruker) with a working frequency
400 MHz for 1H NMR. Chemical shifts, ppm, are given
in δ scale relative to internal standards [Me4Si for
organic solvents and sodium 3-(trimethylsilyl)propane-
EXPERIMENTAL
sulfonate (DSS) for D2O], coupling constants in Hz.
Trimethylbromosilane, diethyl azodicarboxylate,
triphenylphosphine, and triethyl phosphonoformate The working frequency of 162 MHz was used for P
31
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

246
SHIROKOVA et al.
Table 2. Anti-HIV activity of the phosphonates synthesized
General procedure for the preparation of amides
(Va)–(Vd). Phosphonate (IV) (0.2 mmol) was kept for
18 h at room temperature in 25% ammonia (5 ml), in
25% aqueous methylamine (5 ml), or phenylethylamine
(2 ml), or refluxed in morpholine (3 ml) for 3 h. The
solution was evaporated, the residue was dissolved in
water and chromatographed on a DEAE-Toyopearl col-
umn (2 × 18 cm), eluted with a linear gradient of
Com-
pound
CD50, µM ID50, nM ID90, nM
SI
(IIa)
141.7
197.1
67.4
7.1
3.9
9
16
75
20140
50539
7489
(IIb)
(IIc)
47
NH4HCO3 in water (0
0.1 M). The target fractions
(IId)
(IIe)
37.1
6.4
31
5797
were evaporated, the residue was dissolved in water
(1 ml) and chromatographed on a LiChroprep RP-18
column (2 × 18 cm) for (Va) and (Vb) or LiChroprep
RP-8 for (Vc) and (Vd) eluted with water. The UV-
absorbing target fractions were evaporated.
195.5
248.6
103
600
3900
19
326
(IIIa)
(IIIb)
(IIIc)
(IIId)
(IIIe)
(IIIf)
(IIIg)
(IIIh)
7.7
2.6
9.6
2
32286
39623
5000
>2.6
48
28.8
6
3'-Azido-3'-deoxythymidine
5'-aminocarbon-
ylphosphonate (Va); yield 94%; 1H NMR(DMSO-d6):
7.82 (1 H, m, H6), 7.17 (1 H, s, NH2, Ha), 7.13 (1 H, s,
NH2, Hb), 6.12 (1 H, t, J 6.9, H1'), 4.50 (1 H, m, H3'),
3.95 (3 H, m, H4' and H5'), 2.30 (2 H, m, H2'), 1.81 (3
H, br. s, 5-CH3); 31P NMR (DMSO-d6): –1.56 (s); MS,
m/z 374.1 [M]+, Mcalc 374.07.
240.2
69.8
120100
>31727
470417
76777
>60476
<2.2
<220
112.9
69.1
0.24
7.2
2.2
2.1
0.9
127.0
<2.1
AZT
187.5
17
750
11029
3'-Azido-3'-deoxythymidine
5'-(methylami-
1
nocarbonyl)phosphonate (Vb); yield 91%; H NMR
(D2O): 7.52 (1 H, s, H6), 6.06 (1 H, t, J 6.7, H1'), 4.32
(1 H, m, H3'), 4.01 (3 H, m, H4' and H5'), 2.61 (3 H, s,
ëH3NH), 2.32 (2 H, m, H2'), 1.72 (3 H, s, 5-CH3); 31P
NMR (with phosphorus–proton decoupling; chemical
shifts are given relative to 85% H3PO4 as an external
standard). Mass spectra were registered on a COMPACT
MALDI-4 mass spectrometer (Kratos Analytical,
United States).
NMR (D2O): –1.40 (s); MS, m/z: 388.1 [M+], Mcalc
388.09.
3'-Azido-3'-deoxythymidine 5'-ethoxycarbon-
ylphosphonate (IV). Trimethylbromosilane (2 ml,
15.5 mmol) was added to a solution of triethyl
phosphonoformate (1 ml, 5.28 mmol) in CCl4 (2 ml) at
0°C. The reaction mixture was kept for 16 h at 20°C and
evaporated. The residue was coevaporated with toluene
(3 × 10 ml) and treated with 1 : 1 pyridine–water mix-
ture (4 ml). After 1 h, the solution was evaporated,
coevaporated with pyridine (10 ml × 3), dissolved in
pyridine (20 ml), and AZT (0.9 g, 3.37 mmol) and piv-
aloyl chloride (0.6 ml, 4.87 mmol) were added. The
reaction mixture was stirred for 18 h at 6°C, diluted
with water to the volume of 100 ml, and applied onto a
DEAE-Toyopearl column (3 × 20 cm). The product was
eluted with a linear gradient of NH4HCO3 in water
3'-Azido-3'-deoxythymidine 5'-(phenethylami-
nocarbonyl)phosphonate (Vc); yield 86%; H NMR
1
(D2O): 7.42 (1 H, s, H6), 7.10 (5 H, m, C6H5), 6.02
(1 H, t, J 6.5, H1'), 4.17 (1 H, m, H3'), 3.90 (1 H, m,
H4'), 3.81 (2 H, m, H5'), 3.39 (2 H, m, ëH2NH), 2.69
(2 H, t, J 6.5, ëH2C6H5), 2.24 (2 H, m, H2'), 1.68 (3 H,
31
s, 5-CH3); P NMR (D2O): –1.57 (s); MS, m/z: 478.1
[M]+, Mcalc 478.14.
3'-Azido-3'-deoxythymidine
5'-[(1-mor-
1
pholino)carbonyl]phosphonate (Vd); yield 77%; H
NMR (D2O): 7.69 (1 H, s, H6), 6.15 (1 H, t, J 6.5, H1'),
4.48 (1 H, m, H3'), 4.17 (1 H, m, H4'), 4.11 (2 H, m,
H5'), 3.53–3.86 (8 H, 4 m, morpholine), 2.41 (2 H, m,
H2'), 1.79 (3 H, s, 5-CH3); 31P NMR (D2O): –1.43 (s);
(0
0.15 M), and the target fractions were evapo-
MS, m/z: 444.1 [M]+, Mcalc 444.12.
rated and coevaporated with water (10 ml × 5). The res-
idue was diluted with water (2 ml) and chromato-
graphed on a LiChroprep RP-8 column (2.5 × 25 cm)
eluted with water. The UV-absorbing fractions were
evaporated to give 1.2 g (85%) of phosphonate (IV) as
an ammonium salt; 1H NMR (D2O): 7.58 (1 H, m, H6),
6.15 (1 H, t, J 7.5, H1'), 4.4 (1 H, m, H3'), 4.10 (2 H, m,
ëH2ëH3), 4.07 (3 H, m, H4' and H5'), 2.38 (2 H, m,
H2'), 1.78 (3 H, br. s, 5-CH3), 1.11 (3 H, t, J 7.1,
A general procedure for the synthesis of phos-
phonates (II) and (III). MethodA. The corresponding
alcohol (1 ml) was added to a solution of phosphonate
(IV) or (V) (0.2 mmol) in pyridine (3 ml). The mixture
was cooled to 0°C, treated with TPSCl (0.6 mmol), kept
for 16 h at room temperature, and evaporated. The res-
idue was chromatographed on a silica gel column (2 ×
25 cm) eluted with a gradient of methanol in chloro-
31
ëH2CH3); P NMR (D2é): –4.93 (s); MS, m/z 403.1
form (0
10%). The fractions containing the target
[M+], Mcalc 403.09.
(II) or (III) were evaporated to dryness.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

NEW PHOSPHONOFORMIC ACID DERIVATIVES OF 3'-AZIDO-3'-DEOXYTHYMIDINE
247
Method B. PPh3 (0.4 mmol), the corresponding 5-CH3), 1.65 (6 H, m, adamantyl), 1.30 (3 H, m,
ëH2CH3); 31P NMR (CD3CN): –8.68 (s), –9.13 (s);
MS, m/z: 537.2 [M]+, Mcalc 537.20.
alcohol (0.5 ml), triphenylphosphine (0.4 mmol), and
diethyl azodicarboxylate (0.4 mmol) were added to a
solution of phosphonate (IV) or (V) (0.2 mmol) in
DMF (2 ml) at 18°ë. The reaction mixture was stirred
for 16 h at 18°ë, evaporated, and coevaporated with tol-
uene. The residue was chromatographed on a silica gel
column (2 × 25 cm) eluted with a gradient of methanol
3'-Azido-3'-deoxythymidine 5'-ethyl aminocar-
bonylphosphonate (IIIa) was obtained from phospho-
1
nate (V) by method A in 72% yield; H NMR
4
((CD3CN): 9.43 (1 H, s, 3-NH), 7.41 (0.5 H, q, J 1,
H6), 7.39 (0.5 H, q, 4J 1, H6), 7.24 (1 H, br. s, NH2, Ha),
in chloroform (0
10%).
3
6.83 (1 H, d, JP–H 34.9, NH2, Hb), 6.14 (1 H, t, J 6.5,
3'-Azido-3'-deoxythymidine 5'-ethyl ethoxycar-
H1'), 4.41 (1 H, m, H3'), 4.34 (2 H, m, H5'), 4.21 (2 H,
m, ëH2ëç3), 4.03 (1 H, m, H4'), 2.39 (2 H, m, H2'),
1.85 (3 H, m, 5-CH3), 1.33 and 1.32 (3 H, 2 t, J 7,
ëH2CH3); 31P NMR (CD3CN): –0.08 (s), –0.35 (s);
MS, m/z: 402.1 [M]+, Mcalc 402.11.
bonylphosphonate (IIa) was obtained by method A in
1
67% yield; H NMR (CD3CN): 8.98 (1 H, s, 3-NH),
7.43 (1 H, m, H6), 6.27 (1 H, t, J 7.5, H1'), 4.4 (1 H, m,
H3'), 4.31 (4 H, m, ëH2ëH3), 4.2 (2 H, m, H5'), 4.07 (1
H, m, H4'), 2.25 (2 H, m, H2'), 1.88 (3 H, br. s, 5-CH3),
1.57 and 1.52 (6 H, 2 m, ëH2CH3); 31P NMR (CD3CN):
–4.84 (s), –4.37 (s); MS, m/z: 431.1 [M]+, Mcalc 431.12.
3'-Azido-3'-deoxythymidine 5'-methyl ami-
nocarbonylphosphonate (IIIb) was obtained from
phosphonate (V) and methanol by method A in 60%
1
3'-Azido-3'-deoxythymidine 5'-phenethyl ethoxy-
carbonylphosphonate (IIb) was obtained by method
A in 53% yield; 1H NMR (CDCl3): 9.05 (1 H, s, 3-NH),
7.36 (1 H, m, H6), 7.19–7.29 (5 H, m, C6H5), 6.23 (1 H,
t, J 7.5, H1'), 4.47 (2 H, m, H5'), 4.30 (5 H, m, ëH2ëH3,
ëH2OP, H3'), 3.97 (1 H, m, H4'), 3.04 (2 H, t, J 6.8,
ëH2C6H5), 2.25 (2 H, m, H2'), 1.88 (3 H, br. s, 5-CH3),
yield; H NMR (CD3CN): 9.26 (1 H, s, 3-NH), 7.39
(0.5 H, q, 4J 1, H6), 7.38 (0.5 H, q, 4J 1, H6), 7.18 (1 H,
br. s, NH2, Ha), 6.80 (1 H, d, 3JP–H 34.7, NH2, Hb), 6.15
(1 H, t, J 6.5, H1'), 4.43–4.33 (3 H, m, H3' and H5'),
4.03 (2 H, m, H4'), 3.83 (1.5 H, d, J 11, ëH3OP), 3.84
(1.5 H, d, J 11, ëH3OP), 2.38 (2 H, m, H2'), 1.86 (1.5
H, d, 5-CH3), 1.85 (1.5 H, d, 5-CH3), 1.27 (3 H, m,
1.32 (3 H, m, ëH2CH3). 31P NMR (CDCl3): –4.13 (s),
−4.32 (s); MS, m/z: 507.2 [M]+, Mcalc 507.15.
31
ëH2CH3); P NMR (CD3CN): 1.12 (s), 0.84 (s); MS,
m/z: 388.1 [M]+, Mcalc 388.09.
3'-Azido-3'-deoxythymidine 5'-propyl aminocar-
3'-Azido-3'-deoxythymidine 5'-isopropyl ethoxy-
carbonylphosphonate (IIc) was obtained by methodA
in 40% yield; 1H NMR (CD3CN): 9.38 (1 H, s, 3-NH),
7.39 (1 H, m, H6), 6.15 (1 H, m, H1'), 4.85 (1 H, m,
H3'), 4.36 (3 H, m, H5', ëH(CH3)2), 4.30 (2 H, m,
ëH2CH3), 4.06 (1 H, m, H4'), 2.38 (2 H, m, H2'), 1.85
(3 H, br. s, 5-CH3), 1.36 (6 H, d, J 5.9, ëH(CH3)2), 1.31
bonylphosphonate (IIIc) was obtained from phospho-
1
nate (V) and propanol by method B in 58% yield; H
NMR ((CDCl3): 9.08 and 9.06 (1 H, 2s, 3-NH), 7.38–
7.33 (2 H, m, H6, Ha of NH2), 6.49 (1 H, d, 3JP–H 35, Hb
of NH2), 6.17 (0.5 H, t, J 6.5, H1'), 6.16 (0.5 H, t, J 6.5,
H1'), 4.42 (3 H, m, H3', H5'), 4.15 (2 H, m, CH2OP),
4.02 (1 H, m, H4'), 2.41 (2 H, m, H2'), 1.93 (1.5 H, s, 5-
CH3), 1.92 (1.5 H, s, 5-CH3), 1.74 (2 H, m, CH2CH3),
(3 H, m, ëH2CH3); 31P NMR (CD3CN): –4.53 (s),
−4.70 (s); MS, m/z: 445.1 [M]+, Mcalc 445.14.
0.96 (3 H, t, J 7.3, CH2CH3); 31P NMR (CDCl3): –0.80
(s), –1.14 (s); MS, m/z: 416.1 [M]+, Mcalc 416.12.
3'-Azido-3'-deoxythymidine 5'-cyclohexyl ethoxy-
carbonylphosphonate (IId) was obtained by method
3'-Azido-3'-deoxythymidine
5'-[2-(2-methyl)-
1
A in 54% yield; H NMR (CD3ëN): 9.27 (1 H, s,
ethyl] aminocarbonylphosphonate (IIId) was obtained
from phosphonate (Va) and isopropanol by method B in
3-NH), 7.41 (0.5 H, q, 4J 1.2, H6) 7.42 (0.5 H, q, 4J 1.2,
H6), 6.14 (1 H, m, H1'), 4.60 (1 H, m, H3'), 4.38 (3 H,
m, H5', ëH(CH2)2), 4.31 (2 H, m, ëH2CH3), 4.06 (1 H,
m, H4'), 2.39 (2 H, m, H2'), 1.85 (3 H, m, 5-CH3), 1.70–
1.28 (10 H, m, CH2 of cyclohexyl), 1.30 (3 H, m,
1
51% yield; H NMR (CD3CN): 9.01 (1 H, s, 3-NH),
7.41 (0.5 H, q, 4J 1, H6), 7.38 (0.5 H, q, 4J 1, H6), 7.07
3
(1 H, br. s, Ha of NH2), 6.69 (1 H, d, JP–H 35, Hb of
NH2), 6.14 (0.5 H, t, J 6.6, H1'), 6.15 (0.5 H, t, J 6.6,
H1'), 4.79 (1 H, m, H3'), 4.39 (1 H, m, CH(CH3)2), 4.32
(2 H, m, H5'), 4.03 (1 H, m, H4'), 2.38 (2 H, m, H2'),
1.86 (1.5 H, d, 5-CH3), 1.85 (1.5 H, d, 5-CH3), 1.35 (6
H, m, CH(CH3)2); 31P NMR ((CD3CN): −1.07 (s), –
1.37 (s); MS, m/z: 416.1 [M]+, Mcalc 416.12.
ëH2CH3); 31P NMR (CD3CN): –4.74 (s), –4.90 (s);
MS, m/z: 485.1 [M]+, Mcalc 485.17.
3'-Azido-3'-deoxythymidine 5'-adamantyl ethox-
ycarbonylphosphonate (IIe) was obtained by method
1
A in 28% yield; H NMR (CD3ëN): 9.17 (1 H, s,
3'-Azido-3'-deoxythymidine
5'-(2,2-dimethyl-
3-NH), 7.40 (1 H, m, H6), 6.15 (1 H, m, H1'), 4.39 (1 H,
m, H3'), 4.32 (2 H, m, H5'), 4.28 (2 H, m, ëH2CH3),
4.04 (1 H, m, H4'), 2.39 (2 H, m, H2'), 2.22 (3 H, br. s,
adamantyl), 2.14 (6 H, m, adamantyl), 1.85 (3 H, br. s,
propyl) aminocarbonylphosphonate (IIId) was
obtained from phosphonate (Va) and 2,2-dimethylpro-
panol by method B in 54% yield; H NMR (CDCl3):
1
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

248
SHIROKOVA et al.
9.62 (0.5 H, s, 3-NH), 9.59 (0.5 H, s, 3-NH), 7.52–7.40
(2 H, m, H6, Ha of NH2), 6.78 (1 H, d, 3JP–H 35, Hb of
NH2), 6.20 (0.5 H, t, J 6.5, H1'), 6.18 (0.5 H, t, J 6.5,
H1'), 4.45 (3 H, m, H3', H5'), 4.05 (1 H, m, H4'), 3.87
(2 H, m, ëH2ë(CH3)3), 2.45 (2 H, m, H2'), 1.94 (1.5 H,
s, 5-CH3), 1.93 (1.5 H, s, 5-CH3), 0.97 (9 H, m,
The stability of (IIIa) was studied in 0.05 M sodium
phosphate buffer at 37°ë and pH 5.5–7.8; the concen-
tration of phosphonate (IIIa) was 0.45 mM. Samples
(10 µl) were taken out after certain intervals, and 0.1 M
phosphate buffer (20 µl, pH 2.9) was added to stop the
process. The mixture was frozen with liquid nitrogen
and was stored at –18°ë.The reaction mixture compo-
sition was analyzed by HPLC under the conditions
described above.
C(CH3)3); 31P NMR (CDCl3): –0.81 (s), –1.13 (s); MS,
m/z: 444.2 [M]+, Mcalc 444.15.
Determination of antiviral acivity. The MT-4 cells
infected with HIV-1 (strain GKV-4046) was used. The
multiplicity of infection was 0.2–0.5 U/cell. The cells
were cultured in an RPMI-1640 medium containing
10% of fetal serum, 300 µg/ml of L-glutamine,
80 µg/ml of gentamycin, and 30 µg/ml of lincomycin at
37°ë in the atmosphere of 5% CO2. For infection, the
3'-Azido-3'-deoxythymidine 5'-ethyl methylami-
nocarbonylphosphonate (IIIf) was obtained from
phosphonate (Vb) and ethanol by method B in 64%
yield; 1H NMR (CD3CN): 9.47 (1 H, s, 3-NH), 7.72 (1
H, m, CH3NH), 7.43 (1 H, s, H6), 6.22 (1 H, t, J 6.8,
H1'), 4.40 (3 H, m, H3', H5'), 4.22 (2 H, m, ëH2ëH3),
4.01 (1 H, m, H4'), 2.91 (1.5 H, s, CH3N), 2.89 (1.5 H,
s, CH3N), 2.39 (2 H, m, H2'), 1.94 (1.5 H, s, 5-CH3),
cells at the concentration of 2 × 106 U/ml and with via-
bility exceeding 90% were used.
1.93 (1.5 H, s, 5-CH3), 1.37 (3 H, m, ëH2CH3); 31P
NMR (CD3CN): –0.10 (s), –0.54 (s); MS, m/z: 416.1
[M]+, Mcalc 416.12.
Cytotoxicity. MT-4 cells were cultured in the pres-
ence of various doses of the compounds under study
(0.001–100 mg/ml) (three wells for each dilution) in
96-well plates for 3 days. The cell concentrations and
viability were measured using an MTT test [10]. The
concentration causing the death of 50% cell (CD50) was
determined using the plotted dose-dependent curve.
3'-Azido-3'-deoxythymidine 5'-ethyl (phenethyl-
amino)carbonylphosphonate (IIIg) was obtained
from phosphonate (Vc) and ethanol by method A in
1
70% yield; H NMR ((CD3CN): 9.43 (1 H, s, 3-NH),
7.62 (1 H, m, CH2NH), 7.41 (0.5 H, s, H6), 7.40 (0.5 H,
s, H6), 7.25 (5 H, m, C6H5), 6.15 (1 H, t, J 6.8, H1'),
4.33 (1 H, m, H3'), 4.27 (2 H, m, H5'), 4.12 (2 H, m,
ëH2ëH3), 4.00 (1 H, m, H4'), 3.53 (2 H, m, ëH2NH),
2.84 (2 H, t, J 7, ëH2C6H5), 2.36 (2 H, m, H2'), 1.86
(0.5 H, s, CH3), 1.84 (0.5 H, s, CH3), 1.28 (3 H, m,
ëH2CH3); 31P NMR ((CD3CN): 0.04 (s), –0.40 (s); MS,
m/z: 506.2 [M]+, Mcalc 506.17.
Anti-HIV activity. The infected MT-4 cells were
cultured in the presence of various doses of the com-
pounds under study (0.001–100 mg/ml) in 96-well
plates for 3 days. The anti-HIV activity was determined
by immunoassay measuring the amount of p24 antigen
[11]. The plotted dose-depending curve was used for
the calculation of quantitative parameters of the inhibi-
tion of HIV-1 replication.
3'-Azido-3'-deoxythymidine 5'-ethyl (1-mor-
pholino)carbonylphosphonate (IIIh) was obtained
from phosphonate (Vd) and ethanol by method A in
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research, project no. 02-04-49009; by the
State AIDS program, project 31; and the International
Research and Technology Center, project no. 1244.
1
47% yield; H NMR ((CD3CN): 9.43 (1 H, s, 3-NH),
4
7.47 (0.5 H, q, J 1.1, H6), 7.43 (0.5 H, q, J 1.1, H6),
6.15 (1 H, t, J 6.5, H1'), 4.44 (1 H, m, H3'), 4.36 (2 H,
m, H5'), 4.21 (2 H, m, ëH2ëH3), 4.06 (1 H, m, H4'),
3.58–3.89 (8 H, m, morpholine), 2.38 (2 H, m, H2'),
1.84 (3 H, br. s, 5-CH3), 1.33 (3 H, t, J 7, ëH2CH3); 31P
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