New dinucleoside phosphonate derivatives as prodrugs of 3′-Azido-3′-deoxythymidine and β -L-2′,3′-Dideoxy- 3′-Thiacytidine: Synthesis and Anti-HIV Properties

Article abstract of DOI:10.1080/15257770.2013.869340

New phosphonate homodimers of 3′-azido-3′-deoxythymidine (AZT) and a phosphonate heterodimer of β-L-2′,3′-dideoxy-3′- thiacytidine (3TC) and AZT were synthesized. The compounds demonstrated moderate anti-HIV activity. Stability of the compounds in human blood serum was studied. A correlation between anti-HIV activity and stability was defined. 2014 Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2013.869340

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New Dinucleoside Phosphonate
Derivatives as Prodrugs of 3′-Azido-3′-
Deoxythymidine and β-L-2′,3′-
Dideoxy-3′-Thiacytidine: Synthesis and
Anti-HIV Properties
Pavel N. Solyeva, Maxim V. Jaskoa, Inna L. Karpenkoa, Yury A.
Sharkina, Alexander V. Shipitsyna & Marina K. Kukhanovaa
a Engelhardt Institute of Molecular Biology RAS, Moscow, Russia
Published online: 24 Mar 2014.
To cite this article: Pavel N. Solyev, Maxim V. Jasko, Inna L. Karpenko, Yury A. Sharkin, Alexander V.
Shipitsyn & Marina K. Kukhanova (2014) New Dinucleoside Phosphonate Derivatives as Prodrugs of 3′-
Azido-3′-Deoxythymidine and β-L-2′,3′-Dideoxy-3′-Thiacytidine: Synthesis and Anti-HIV Properties,
Nucleosides, Nucleotides and Nucleic Acids, 33:2, 64-79, DOI: 10.1080/15257770.2013.869340
To link to this article: http://dx.doi.org/10.1080/15257770.2013.869340
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Nucleosides, Nucleotides and Nucleic Acids, 33:64–79, 2014
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.869340
NEW DINUCLEOSIDE PHOSPHONATE DERIVATIVES
AS PRODRUGS OF 3ꢀ-AZIDO-3ꢀ-DEOXYTHYMIDINE
AND β-L-2ꢀ,3ꢀ-DIDEOXY-3ꢀ-THIACYTIDINE: SYNTHESIS
AND ANTI-HIV PROPERTIES
Pavel N. Solyev, Maxim V. Jasko, Inna L. Karpenko, Yury A. Sharkin,
Alexander V. Shipitsyn, and Marina K. Kukhanova
Engelhardt Institute of Molecular Biology RAS, Moscow, Russia
GRAPHICAL ABSTRACT
O
O
P
R
O
P
O
O
O
Cyt
Thy
Thy
Thy
O
O
O
O
CH2
S
OPh
N3
N3
2a R = -Ph,
N3
2d R = -CH2F,
3
2b R = -CH2OCH3, 2e R = -CH2I,
2c R = -CH2OPh,
2f R = -CH2N3
New phosphonate homodimers of 3ꢁ-azido-3ꢁ-deoxythymidine (AZT) and a phosphonate het-
2
erodimer of β-L-2ꢁ,3ꢁ-dideoxy-3ꢁ-thiacytidine (3TC) and AZT were synthesized. The compounds
demonstrated moderate anti-HIV activity. Stability of the compounds in human blood serum was
studied. A correlation between anti-HIV activity and stability was defined.
Keywords Antiviral nucleosides; HIV nucleosides; nucleoside phosphonates; modified
nucleosides
INTRODUCTION
Nucleoside analogues are widely used for the treatment of HIV infec-
tion. After penetration into cells, nucleoside analogues undergo three sub-
sequent steps of phosphorylation by cellular kinases, followed by integration
of their triphosphates into the 3ꢁ end of the growing proviral DNA being
Received 27 August 2013; accepted 20 November 2013.
The work was supported by the Russian Foundation for Basic Research, projects 12-04-00581-a and
13-04-00829-a; Presidium of the Russian Academy of Sciences, project “Molecular and Cellular Biology.”
Address correspondence to Pavel N. Solyev, Engelhardt Institute of Molecular Biology RAS, 32
Vavilov Street, Moscow, 119991, Russia. E-mail: solyev@gmail.com
64

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
65
catalyzed by reverse transcriptase and terminate its synthesis. Among the
nucleoside-based derivatives, AZT (RetrovirꢀR ) and 3TC (LamivudineꢀR ) are
the most commonly applied drugs in anti-HIV “cocktails” that usually con-
tain these two HIV reverse transcriptase inhibitors (CombivirꢀR ) alongside
with one integrase or protease inhibitor. Despite progress in the treatment
of HIV-infected patients, these drugs possess some drawbacks: AZT lifetime
lasts in patients only 1 hour,[1] frequent dose administration and long-term
usage of AZT cause toxic side effects, i.e., anemia, bone marrow suppres-
sion, neuropathy and the emergence of HIV resistant strains.[2–4] There
have been developed various depot forms of nucleosides and nucleotides
in order to reduce toxic effects of anti-HIV drugs, as well as to increase
their oral bioavailability or to improve pharmacokinetic properties.[5–7]
A
depot form, being inactive on its own, is converted under chemical or en-
zymatic hydrolysis into an appropriate antiviral inhibitor. Most of depot
forms employ the 5ꢁ position for modification of nucleoside/nucleotide
analogues. Out of a large number of constructed depot forms of HIV drugs,
only one compound was approved by the U.S. FDA for the treatment of HIV
infected patients—the prodrug of tenofovir (VireadꢀR ).[5,8] Another depot-
form—a prodrug of AZT—(5ꢁ-hydrogen-phosphonate AZT, NikavirꢀR ) was
approved in Russia for the prevention and treatment of AIDS.[9–11] Phos-
phate/phosphonate derivatives of homo- and heterodimers of anti-HIV nu-
cleoside drugs present a special interest. Such derivatives are depot forms of
one or two HIV inhibitors joined together in one molecule. If hydrolyses of
components occur at different time or with different rates, the lifetime of ac-
tive drugs in vivo may be prolonged. Furthermore, two nucleoside anti-HIV
drugs can give a synergistic effect that allows us to reduce concentrations of
the drugs.[12] The first anti-HIV compounds of this kind, namely bis-(3ꢁ-azido-
3ꢁ-deoxythymidin-5ꢁ-O-yl) methylphosphonate and bis-(2ꢁ,3ꢁ-dideoxycytidin-
5ꢁ-O-yl) methylphosphonate, were obtained in 1990.[13] However, they were
not readily hydrolyzed to form the active components and did not show
anti-HIV activity. Later on, bis-AZT α-hydroxyphosphonates were reported
to demonstrate anti-HIV activity comparable to that of AZT.[14] It was also
shown that heterodimer conjugates of FLT and AZT with dicarboxylic acids
possessed higher anti-HIV activity compared to the parent FLT and AZT.[15]
Dinucleoside derivatives proved to be active against other viruses as well.
For example, acyclovir phosphate dimer suppressed herpes simplex virus
(HSV-1) replication in cell culture more effectively than acyclovir and had
better bioavailability due to higher solubility than acyclovir.[16] Bis-(3ꢁ-deoxy-
nucleoside) phosphoramidates were shown to be active inhibitors of hepatitis
C virus (HCV) in replicon system.[17] We decided to investigate new dinucle-
oside phosphonates of AZT as prodrugs and make a coupled form of AZT
and 3TC phosphonate prodrug. We aimed to define such a phosphonate
substituent, which would allow one nucleoside ester bond to be hydrolyzed
fast enough to reach the effective drug concentration in blood, while another

66
P. N. Solyev et al.
nucleoside of the phosphonate, being released slowly and gradually, could
maintain the drug concentration in blood for a prolonged period of time.
Earlier, we synthesized different phosphonates of AZT and evaluated their
anti-HIV activity in the cell culture.[18] Apart from the successful NikavirꢀR ,
another phosphonate derivative, AZT aminocarbonylphosphonate, showed
lower toxicity and had improved pharmacokinetics as compared to AZT and
Nikavir.[19] Now this compound is in Phase II clinical trials.[7] Continuing
the search for depot forms of anti-HIV drugs, we focused our attention on
dinucleoside phosphonate derivatives.
Herein, we describe the synthesis of six new phosphonate AZT homod-
imers and one heterodimer containing AZT and 3TC. The stability of com-
pounds in human blood serum, their anti-HIV activity, and toxicity in CEM-
SS cell culture have been studied.
RESULTS AND DISCUSSION
Synthesis and Characterization
Synthesis was performed as described below (Scheme 1). 3ꢁ-Azido-3ꢁ-
deoxythymidin-5ꢁ-O-yl derivatives were obtained by three different methods:
by the reaction of the two-fold excess of AZT with the corresponding phos-
phonic dichloride (this method was used to produce 2a–b); or by the direct
condensation of the corresponding phosphonic acid with the two-fold excess
of the nucleoside and TPS-Cl (synthesis of 2c–e); as well as by the reaction
of AZT with 3ꢁ-azido-3ꢁ-deoxythymidin-5ꢁ-O-yl -azidomethylphosphonate 1b
and DCC as a condensing agent in the case of 2f synthesis.
Dinucleoside heterodimer 3 synthesis required step-by-step condensa-
tion of phenoxymethylphosphonic acid with equimolar amount of AZT to
afford phosphonate 1c followed by its reaction with 3TC in the presence
of TPS-Cl. We did not protect amino group of 3TC since its further depro-
tection would cleave the 5ꢁ,5ꢁ-phosphonodiester bond. A minor product of
coupling with 3TC 4-amino group was also observed and was separated by
column chromatography. It was noticed that 4-NH-product was unstable and
in a week decomposed into starting 3TC and 1c.
This two step route is also feasible for the synthesis of bis-AZT phos-
phonates 2 instead of three methods described above and was used to
obtain azidomethyl-phosphonate 2f. We intended to synthesize 2f by the
substitution of the iodide adjacent to the methylene fragment by azide.
It did not appear to form the desired product in bis-AZT derivatives, but
mono-AZT iodomethylphosphonate 1a successfully substituted the iodide
1
by the azide. This has been proved by the H NMR spectra: protons of the
methylene group in the obtained 1b are equivalent and the doublet with
2J
= 11.2 Hz is observed, while 1a protons of the methylene group are
CH2, P

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
67
SCHEME 1 Synthesis of homodimers of AZT (2) and a heterodimer of AZT and 3TC (3).
non-equivalent and demonstrate a more complex ABX system with the gem-
inal proton–proton coupling constant of CH2–P fragment 2J
= 12.8 Hz
Ha, Hb
and geminal constants 2J
of 9.3 and 9.0 Hz (Figure 1).
H, P
Similar azidation of bis-AZT derivatives (1e → 1f) led to the side process
of AZT 5ꢁ,5ꢁ-phosphonodiester bond cleavage.
1
1
Determination of J
and J
in 13C NMR spectrum of 3ꢁ-azido-3ꢁ-
C, F
C, P
deoxythymidin-5ꢁ-O-yl fluoromethylphosphonate 2d was rather complicated
due to their close values, but the 13C satellites in 31P NMR allowed to as-
1
sign J
unambiguously. Additionally, 2d was characterized by 19F NMR
P, C
confirming all the other spin-coupling constants (Figure 2).
All the products were obtained in good yields and fully characterized by
NMR spectra. Their λmax in UV spectra corresponded to those for thymidine
derivatives (267 nm), heterodimer 3 displayed λmax of intermediate position
(experimental: 268 nm) for thymidine (267 nm) and cytidine (271 nm)
derivatives. Starting phosphonic acids were commercial or synthesized by
the reported standard procedures.[20–23]
Anti-HIV Activity and Toxicity
Anti-HIV activity and toxicity of the compounds were estimated in
HIV-1BRU infected CEM-SS (human T4-lymphoblastoid) cell line as de-
scribed earlier.[24] The anti-HIV activity of the compounds (Table 1) de-
pends on the phosphonate moiety structure. Homodimer 2d appeared to

68
P. N. Solyev et al.
FIGURE 1 X-CH2-P Peaks in 1H NMR of 1a and 1b. (Color figure available online).
be the most active compound. Its inhibiting activity was twice higher than
that of AZT and eight times higher than Nikavir. Heterodimer 3, containing
both AZT and 3TC, did not reveal any advantages over AZT and the rest of
AZT homodimers demonstrated lower anti-HIV activity than the reference
drugs. It is important to mention the augmented toxicity of the synthesized
dinucleosides. Product 2d was about five and six times more toxic than AZT
TABLE 1 Anti-HIV activity of homo- and heterodimers in CEM-SS cell culture
Compound
EC50a (μM)
CC50b (μM)
SIc
2a
2b
2c
2d
2e
2f
3
Ph-P(O)(AZT)2
CH3OCH2-P(O)(AZT)2
PhOСH2-P(O)(AZT)2
FCH2-P(O)(AZT)2
ICH2-P(O)(AZT)2
N3CH2-P(O)(AZT)2
PhOCH2-P(O)(AZT)(3TC)
AZT
10
33
31
37
29
39
24.5
60
142
185
3.3
46
148
1933
3.5
136
250
3837
1405
0.68
0.25
0.015
11
0.18
0.24
0.037
0.131
Nikavir
aEC50, effective concentration of compounds required to inhibit 50% of viral replication.
bCC50 , cytotoxic concentration of compounds required to inhibit 50% CEM-SS cell growth.
cSI, selectivity index, CC50/ EC50
.

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
69
1
1
FIGURE 2 Assignments of J
and J
in 13C NMR proton decoupled spectrum of 2d (a) via the
C, F
C, P
13C satellites in 31P NMR proton decoupled spectrum (b); 19F NMR non-decoupled (c) and proton
decoupled (d) spectra of 2d. (Color figure available online).
and Nikavir, respectively; as a result, its selectivity index was twice lower than
that of AZT.
Anti-HIV properties of corresponding mono-AZT phosphonates (FCH2-
P(O)(OH)(AZT), ICH2-P(O)(OH)(AZT) and N3CH2-P(O)(OH)(AZT))
bearing the same substituents as the synthesized bis-AZT phosphonates 2d–f
were studied in our laboratory and described earlier.[18]
The comparison of mono-AZT and bis-AZT phosphonates revealed that
SI of 2d was more than one order higher than that of FCH2-P(O)(OH)(AZT)
(1933 vs. 150), while SI for 2e was much lower than that of ICH2-
P(O)(OH)(AZT) (3.5 vs. 24). Phosphonate 2f possessed rather poor SI,
as well as its mono-AZT derivative N3CH2-P(O)(OH)(AZT) (136 vs. 107).

70
P. N. Solyev et al.
FIGURE 3 HPLC separation of the hydrolysis products of 2d and 3 after incubation with human blood
serum. Products of compound 2d after 50 min incubation (a); products of compound 3 after 12 hours
incubation (b).
Thus, antiviral properties of the anionic mono-AZT phosphonates not always
correlate with that of the corresponding bis-AZT phosphonates, and their
phosphonate moiety structure and antiviral activity relationship is rather
complex to predict it ahead of their synthetic realization into a phospho-
nate depot form.
Differences in antiviral properties of dinucleoside monophosphates
and corresponding mononucleotide analogues were observed earlier.[12]
It was noticed that AZT homodimer (AZT)P(O)(OH)(AZT) was more
toxic than heterodimers consisting of two anti-HIV drugs. Besides, toxic-
ity varies depending on cell cultures used. The pharmacokinetic study of
(AZT)P(O)(OH)(PMPA) allowed to infer that this compound was a depot
form gradually releasing AZT and PMPA, and had lower toxicity. Neverthe-
less, its antiviral activity in cell cultures did not exceed the overall activity of
AZT and PMPA combination.[25]
Hydrolysis in Human Blood Serum
Anti-HIV activity of depot forms depends on their hydrolysis rates and
the conversion into an active drug form. Therefore, we evaluated the chem-
ical stability of dinucleoside derivatives and their stability in human blood
serum.
All the studied compounds were chemically stable for more than a day
in pH range 4.5–7. Hydrolysis of the homodimers (2a–f) in blood serum
resulted in the mixture of two products: AZT and AZT monophosphonate.
At the same time, hydrolysis of the heterodimer 3 resulted in the mixture
of four products: AZT and 3TC monophosphonate, as well as 3TC and AZT

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
71
TABLE 2 Stability of the compounds in human blood serum
Compound
T1/2 (hours)
2a
2b
2c
2d
2e
2f
3
Ph-P(O)(AZT)2
CH3OCH2-P(O)(AZT)2
PhOСH2-P(O)(AZT)2
FCH2-P(O)(AZT)2
ICH2-P(O)(AZT)2
N3CH2-P(O)(AZT)2
PhOCH2-P(O)(AZT)(3TC)
Nikavir
>24
>24
>24
0.78
>24
12
21
6
monophosphonates. The obtained products were separated by HPLC, the
retention times for starting and obtained compounds are given in the “Ma-
terials and Methods” section. Half-life data (T1/2) were calculated according
to the peak area at a different time of hydrolysis. An example of the product
separation for 3 is presented in Figure 3.
Half-life data (T1/2) of the compounds in human blood serum are shown
in Table 2. We think that the hydrolysis of the compounds in human blood
serum depends on cellular enzymes such as 5ꢁ-nucleotidases or other hy-
drolyzing enzymes that are mentioned in many publications.[26]
A correlation between hydrolysis half-life and anti-HIV activity of the
synthesized dimers is evident (Tables 1 and 2). The compounds showing
lower activity appeared to be more stable in human blood serum. All this
suggest that the leading anti-HIV activity of 2d can be explained by its fast
hydrolysis to the parent nucleoside.
CONCLUSIONS
To sum up, we have designed and synthesized new phosphonate deriva-
tives with different phosphonate moieties containing two nucleoside drugs in
a single molecule and studied their anti-HIV activity, toxicity, and hydrolysis
rates. The results confirm that the dimers acted as depot forms. Their antivi-
ral activity in cell cultures was comparable to the referent AZT and NikavirꢀR ,
and even surpassed them twice for bis-(3ꢁ-azido-3ꢁ-deoxythymidin-5ꢁ-O-yl)
fluoromethylphosphonate 2d. But the increased toxicity demonstrated in
CEM-SS cells remains a problem to be solved.
EXPERIMENTAL
Materials and Methods
Reagents and solvents were purchased from Acros (Belgium). The fol-
lowing starting phosphonic compounds were synthesized according to the

72
P. N. Solyev et al.
procedures previously described in the literature: methoxymethylphospho-
nic dichloride,[20] iodomethylphosphonic acid,[21] phenoxymethylphospho-
nic acid,[22] and fluoromethylphosphonic acid.[23] AZT and 3TC were a kind
gift of the “AZT Association” (Moscow, Russia). The solvents were purified
by standard procedures.
NMR spectra of the products (δ, ppm; J , Hz) were registered on an AMX
III-400 spectrometer (Bruker BioSpin Gmbh, Karlsruhe, Germany) with the
working frequencies of 400 MHz for 1H NMR, 162 MHz for 31P NMR (with
P–H interaction decoupling) and 100.6 MHz for 13C NMR (with C–H inter-
action decoupling). 19F NMR spectra (δ, ppm; J , Hz) were registered on an
Avance II-300 spectrometer (Bruker BioSpin Gmbh, Karlsruhe, Germany),
with 282.4 MHz working frequency.
UV spectra were registered on a Shimadzu UV-2401PC spectropho-
tometer (Shimadzu Corporation, Japan) in methanol, in the range
of 200–300 nm and were typical to those of thymidine and cytidine
derivatives.
Mass spectra of the products (m/z,% rel. int.) were registered on an
LCMS-2020 single quadrupole liquid chromatograph mass spectrometer
(Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), using ESI. The mea-
surements were acquired in a positive ion mode (interface capillary voltage,
4800 V), a syringe injection was used for solutions in acetonitrile (flow rate
200 μl/min). Nitrogen was applied as a dry gas; interface temperature was
set at 350◦С.
The reaction process was checked using TLC on precoated Kiesel-
gel 60F254 plates (Merck, Germany) in the following systems: chloro-
form/methanol 19:1, chloroform/methanol 9:1 and dioxane/ammonia
(25% aq.) 4:1. Column chromatography was performed on ion-exchange
DEAE-Toyopearl 650 M resin (Tosoh Corporation, Japan) and on silica gel
(40–63 μm; Merck, Germany).
Anti-HIV activity and toxicity assays. Anti-HIV activity of the tested com-
pounds (0.01–100 μg/ml, three replicates for each dose) was assessed
in CEM-SS cell culture by the measurement of p24 antigen amount us-
ing immunoassay after 3 days of incubation. Cellular cytotoxicity was esti-
mated after incubation of CEM-SS cell in the presence of the compounds
(0.01–500 μg/ml, three replicates for each dose) after 4 days incubation.
The concentration and viability of the cells were measured by the trypan
blue day exclusion colorimetric assay, and the CC50 for each compound was
calculated.[24]
Stability in human blood serum. Tested compounds were incubated
with human blood serum at 37◦C in the concentration of 500 μM. After
certain time intervals, aliquots were taken, and the reaction was terminated
by the addition of cool methanol up to 66% (v/v). The formed precip-
itate was pelleted by centrifugation for 15 min at 20,000×g. The super-
natant was dried in a Speed-Vac concentrator, the residue was dissolved

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
73
in 5 mM aqueous phosphate buffer (pH 5.20), and the products were an-
alyzed by HPLC. The compound concentrations were calculated by peak
areas.
Centrifugation was performed on an Eppendorf 5415 centrifuge (Ep-
pendorf AG, Hamburg, Germany). An automatic Speed-Vac Concentrator
AS260 (Savant, Osterville, MA, USA) was used for concentrating the sample.
HPLC analysis was performed using a Gilson chromatograph (Gilson
Inc., Middleton, WI, USA) supplied with a digital GSIOC 506 controller and a
Gilson-315 UV detector with varying wavelengths. Compounds were detected
at λ 267 nm using a Nucleosil 100 C-18 column (5 μm, 4 × 150 mm) with a
C-18 precolumn (10 μm, 4 × 8 mm); using mobile phase of 80% aqueous
EtOH (A) in 5 mM aqueous phosphate buffer (pH 5.2). Gradient parame-
ters: 0% A for 5 min; 0% → 15% A for 5 min; 15% → 30% A for 10 min; 30%
→ 70% A for 20 min; 70% → 100% A for 5 min; 100% → 0% A for 5 min;
0% A for 5 min; the flow rate of 0.5 ml/min. Retention times were as follows:
AZT, 19.8 min; 3TC, 15.3 min; 2a, 36.7 min; 2b, 29.0 min; 2d, 29.7 min;
2e, 34.0 min; 2f, 32.0 min; 3, 32.0 min; Ph-P(O)(OH)(AZT), 23.8 min;
CH3OCH2-P(O)(OH)(AZT), 16.3 min; PhOCH2-P(O)(OH)(AZT),
25.7 min; PhOCH2-P(O)(OH)(3TC), 20.6 min; FCH2-P(O)(OH)(AZT),
16.7 min; ICH2-P(O)(OH)(AZT), 20.3 min; N3CH2-P(O)(OH)(AZT),
17.8 min.
Standard Procedure for Synthesis of Anionic Phosphonate
Derivatives 1a, 1c
Mono-AZT phosphonates 1a, 1c were synthesized similarly to the previ-
ously studied AZT alkylphosphonates.[27] A solution of the corresponding
phosphonic acid (0.6 mmol) and AZT (0.6 mmol) in pyridine (3 ml) was
treated with DCC (1.2 mmol). The reaction mixture was stirred at room
temperature for 10 hours and then diluted with water. After 30 minutes of
additional stirring the solution was separated from the precipitate, concen-
trated by evaporation under vacuum, and re-diluted with water. The solution
was applied onto a DEAE-Toyopearl column (20 × 175 mm) and eluted with
a linear gradient of NH4HCO3 (0 → 0.2 M, 500 ml). The target fraction was
concentrated by evaporation under vacuum, the residue was diluted with
water and re-evaporated.
Standard Procedure for Synthesis of Dinucleoside Phosphonate
Derivatives 2 and 3
Method A
The following method was used for synthesis of 2a, 2b. AZT (106 mg,
0.4 mmol) was added to a solution of the corresponding phosphonic dichlo-
ride (0.2 mmol) in triethylphosphate (1 ml). The reaction mixture was

74
P. N. Solyev et al.
stirred at 4◦C for 18 h and then diluted with water, concentrated by evapo-
ration under vacuum, applied onto a silica gel column (20 × 250 mm) and
eluted with a gradient of methanol in chloroform (0 → 15%). The target
fraction was concentrated by evaporation under vacuum.
Method B
The following method was used for synthesis of 2c–2e. AZT (106 mg,
0.4 mmol) was added to a solution of the corresponding phosphonic acid
(0.2 mmol) in pyridine (3 ml), cooled to 0◦C and treated with TPS-Cl
(182 mg, 0.6 mmol). The reaction mixture was stirred at 4◦C for 18 hours
and then 1 ml of the saturated aqueous NaHCO3 solution was added. The
mixture was concentrated by evaporation under vacuum, applied onto a sil-
ica gel column (20 × 250 mm) and eluted with a gradient of methanol in
chloroform (0 → 15%). The target fraction was concentrated by evaporation
under vacuum.
Method C
The following method was used for synthesis of 2f and 3. To a solution
of the phosphonate 1 (0.2 mmol) in pyridine (3 ml), an equimolar amount
of the corresponding nucleoside was added (53 mg, 0.2 mmol of AZT for
synthesis of homodimer 2f; or 46 mg, 0.2 mmol of 3TC for synthesis of
heterodimer 3), cooled to 0◦C and treated with TPS-Cl (91 mg, 0.3 mmol).
The reaction mixture was stirred at 4◦C for 18 hours and then 1 ml of the
saturated aqueous NaHCO3 solution was added. The mixture was concen-
trated by evaporation under vacuum, applied onto a silica gel column (20 ×
250 mm) and eluted with a gradient of methanol in chloroform (0 → 15%).
The target fraction was concentrated by evaporation under vacuum.
3’-Azido-3’-deoxythymidin-5’-O-yl -iodomethylphosphonate (1a)
1
Obtained 282 mg (yield 73%) as a yellowish viscous liquid. H NMR, δ
3
(CD3OD): 7.55 (1H, s, H-6), 6.06 (1H, t, J
6.5 Hz, H-1’), 4.36 (1H,
1’, 2’
2
m, H-3’), 3.95–4.02 (3H, m, H-4’ & H-5’), 3.31 (1Н, dd, J
12.8 Hz,
Ha, Hb
2J H , P 9.0 Hz, CHa-P) 3.29 (1H, dd, 2J
12.8 Hz, 2J H , P 9.3 Hz, CHb-P),
Hb, Ha
2.34 (2Н, m, Н-2’), 1.75 (3H, s, 5-CH3). 31P NMR, δ (CD3bOD): 17.00 (s).
a
3’-Azido-3’-deoxythymidin-5’-O-yl -azidomethylphosphonate (1b)
A solution of 1a (94 mg, 0.2 mmol) in DMF (2 ml) was treated with
NaN3 (20 mg, 0.3 mmol). The reaction mixture was stirred at 60◦C for
12 hours, cooled to 0◦C and diluted with water, concentrated by evaporation
under vacuum and re-diluted with water. The solution was applied onto a
DEAE-Toyopearl column (20 × 175 mm) and eluted with a linear gradient
of NH4HCO3 (0 → 0.2 M, 500 ml). The target fraction was concentrated

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
75
by evaporation under vacuum, the residue was diluted with water and re-
evaporated to result in 69 mg (yield 90%) of 1b as a yellowish viscous liquid.
3
1H NMR, δ (CD3OD): 7.58 (1H, s, H-6), 6.11 (1H, t, J 1’, 2’ 6.5 Hz, H-1’),
4.38 (1H, m, H-3’), 4.05 (3H, m, H-4’ & H-5’), 3.33 (2H, d, 2J CH , P 11.2 Hz,
CH2-P), 2.38 (2Н, m, Н-2’), 1.80 (3H, s, 5-CH3). 31P NMR, δ (CD32OD): 17.21
(s).
3’-Azido-3’-deoxythymidin-5’-O-yl -phenoxymethylphosphonate (1c)
Obtained 223 mg (yield 85%) as a colorless viscous liquid. 1H NMR, δ
(CD3OD): 7.82 (1H, q, 4J
m, m-Ph) 6.92 (1H, m, p-Ph), 6.22 (1H, dd, J
H-1’), 4.50 (1H, m, H-3’), 4.22 (2H, m, H-5’), 4.13 (2H, d, J
1.2 Hz, H-6), 7.25 (2H, m, o-Ph), 6.96 (2H,
H-6, 5-CH3
3
3
6.0 Hz, J
7.7 Hz,
10.0 Hz,
1’, 2’a
1’, 2’b
2
H, P
4
CH2-P), 4.09 (1H, m, H-4’), 2.41 (2Н, m, Н-2’), 1.86 (3H, d, J
5-CH3, H-6
1.2 Hz, 5-CH3). 31P NMR, δ (CD3OD): 16.33 (s).
Bis-(3’-azido-3’-deoxythymidin-5’-O-yl) phenylphosphonate (2a)
Obtained 85 mg (yield 65%) as a colorless viscous liquid. 1H NMR, δ
(CDCl3): 10.26 & 10.23 (2H, 2 × s, 3-NH), 7.82 (2H, m, о-Ph), 7.61 (1H, t,
3J p-Ph, m-Ph 7.5 Hz, p-Ph), 7.49 (2H, m, m-Ph), 7.32 & 7.17 (2H, 2 × s, H-6),
6.13 & 6.07 (2H, 2 × t, 3J
6.5 Hz, H-1’), 4.35 (6H, m, H-3’ & H-5’), 4.06
1’, 2’
(2Н, m, H-4’), 2.41 (4Н, m, Н-2’), 1.79 & 1.77 (6H, 2 × s, 5-CH3). 13С NMR,
δ (CDCl3): 164.29 (s, C-4), 150.65 (s, C-2), 135.92 (s, C-6), 133.90 (s, p-Ph),
131.83 (d, 2J
10.4 Hz, o-Ph), 129.12 (d, 3J
16.1 Hz, m-Ph), 126.17 (d,
C, P
C, P
1J C, P 189.5 Hz, i-Ph), 111.46 & 111.38 (2 × s, C-5), 85.82 (2 × s, C-1’), 82.47
(d, 3J C, P 5.6 Hz, C-4’), 65.64 & 65.68 (d, 2J C, P 4.8 Hz, C-5’), 60.79 & 60.69 (2
× s, C-3’), 37.25 & 37.12 (2 × s, C-2’), 12.40 (s, 5-CH3). 31P NMR, δ (CDСl3):
15.91 (s). ESI MS, m/z (% rel. int.): 680.2 (M + Na++H+, 37.0%), 679.3 (M
+ Na+, 100%), 658.4 (M + 2 × H+, 34.4%), 657.3 (M + H+, 25.4%).
Bis-(3’-azido-3’-deoxythymidin-5’-O-yl) methoxymethylphosphonate (2b)
Obtained 87 mg (yield 70%) as a colorless viscous liquid. 1H NMR, δ
4
(DMSO-d6): 10.99 (2H, br.s, 3-NH), 7.50 & 7.47 (2H, 2 × q, J
H-6, 5-CH3
1.2 Hz, H-6), 6.12 (2H, t, 3J 1’, 2’ 6.7 Hz, H-1’), 4.44 (2H, m, H-3’), 4.25 (4H,
2
m, H-5’), 3.99 (2Н, m, H-4’), 3.88 (1H, dd, J
15.5 Hz, 2J
8.0 Hz,
Ha, Hb
Ha, P
CHa-P), 3.86 (1H, dd, 2J
15.5 Hz, 2J
8.0 Hz, CHb-P), 3.35 (3H, d,
Hb, Ha
Hb, P
4J
1.1 Hz, OCH3), 2.46–2.29 (4Н, m, Н-2’), 1.78 (6H, br.s, 5-CH3). 13С
H, P
NMR, δ (DMSO-d6): 163.54 (s, C-4), 150.28 (s, C-2), 135.81 (s, C-6), 109.85
(s, C-5), 83.72 (s, C-1’), 81.42 & 81.38 (2 × d, 3J
5.4 Hz, C-4’), 65.22 (d,
3.1 & 4.5 Hz, C-5’),
C, P
2
1J
162.5 Hz, CH2-P), 65.15 & 65.08 (2 × d, J
C, P
C, P
60.42 (d, 3J C, P 13.0 Hz, OCH3), 59.98 & 59.96 (2 × s, C-3’), 35.50 & 35.46 (2
× s, C-2’), 11.91 & 11.87 (2 × s, 5-CH3). 31P NMR, δ (DMSO-d6): 25.62 (s).
ESI MS, m/z (% rel. int.): 647.2 (M + Na+, 100%), 625.2 (M + H+, 13.9%).

76
P. N. Solyev et al.
Bis-(3’-azido-3’-deoxythymidin-5’-O-yl) phenoxymethylphosphonate (2c)
1
Obtained 103 mg (yield 75%) as a colorless viscous liquid. H NMR,
4
δ (DMSO-d6): 11.30 (2H, br.s, 3-NH), 7.51 & 7.47 (2H, 2 × q, J
H-6, 5-CH3
1.2 Hz, H-6), 7.27 (2H, m, o-Ph), 6.96 (3H, m, m-Ph & p-Ph), 6.13 & 6.11
3
2
(2H, 2 × t, J
7.5 Hz, H-1’), 4.55 (2H, d, J
9.9 Hz, CH2-P), 4.45
1’, 2’
CH2, P
(2H, m, H-3’), 4.33 (4H, m, H-5’), 4.02 (2H, m, H-4’), 2.37 (4Н, m, Н-2’),
1.74 & 1.72 (6H, 2 × d, 4J 5-CH , H-6 1.0 & 1.3 Hz, 5-CH3). 13С NMR, δ (DMSO-
3
d6): 163.55 (s, C-4), 158.07 (d, 3J
14.2 Hz, i-Ph), 150.29 (s, C-2), 135.84
C, P
& 135.80 (2 × s, C-6), 129.43 (s, m-Ph), 121.58 (s, p-Ph), 114.38 (s, o-Ph),
3
109.90 (s, C-5), 83.76 (s, C-1’), 81.41 & 81.35 (2 × d, J
5.8 Hz, C-4’),
C, P
1
65.68 (d, 2J
6.1 Hz, C-5’), 61.00 (d, J
166.5 Hz, CH2-P), 60.01 (s,
C, P
C, P
C-3’), 35.52 (s, C-2’), 11.92 & 11.88 (2 × s, 5-CH3). 31P NMR, δ (DMSO-d6):
24.12 (s). ESI MS, m/z (% rel. int.): 710.2 (M + Na++H+, 33.1%), 709.2 (M
+ Na+, 100.0%), 688.2 (M + 2 × H+, 25.4%), 687.2 (M + H+, 47.9%).
Bis-(3’-azido-3’-deoxythymidin-5’-O-yl) fluoromethylphosphonate (2d)
Obtained 82 mg (yield 67%) as a colorless viscous liquid. 1H NMR, δ
4
(DMSO-d6): 11.01 (2H, br.s, 3-NH), 7.48 & 7.45 (2H, 2 × q, J
H-6, 5-CH3
0.8 Hz, H-6), 6.13 (2H, dd + t, 3J
6.7 Hz, 3J
6.5 Hz; 3J
6.7 Hz,
1’, 2’
1’, 2’a
1’, 2’b
2
2
H-1’), 5.00 (2H, dd, J
45.8 Hz, J
4.5 Hz, CH2-P), 4.45 (2H, m,
CH2, F
CH2, P
H-3’), 4.32 (4H, m, H-5’), 4.01 (2Н, m, H-4’), 2.47–2.30 (4Н, m, Н-2’), 1.78
(6H, br.s, 5-CH3). 13С NMR, δ (DMSO-d6): 163.55 (s, C-4), 150.28 (s, C-2),
135.82 & 135.78 (2 × s, C-6), 109.90 (s, C-5), 83.80 & 83.72 (2 × s, C-1’),
3
1
1
81.26 & 81.20 (2 × d, J
5.4 Hz, C-4’), 76.01 (dd, J
165.2 Hz, J
C, P C, F
C, P
175.9 Hz, CH2-P), 65.56 (br.s, C-5’), 59.85 (s, C-3’), 35.51 & 35.45 (2 × s,
C-2’), 11.88 & 11.84 (2 × s, 5-CH3). 31P NMR, δ (DMSO-d6): 21.25 (d, 2J
P, F
62.6 Hz). 19F NMR, δ (DMSO-d6): -250.53 (dt, 2J F, P 62.6 Hz, 2J F, H 45.8 Hz).
ESI MS, m/z (% rel. int.): 635.3 (M + Na+, 100%), 613.2 (M + H+, 27.5%).
Bis-(3’-azido-3’-deoxythymidin-5’-O-yl) iodomethylphosphonate (2e)
Obtained 101 mg (yield 70%) as a colorless viscous liquid. 1H NMR, δ
(DMSO-d6): 11.24 (2H, s, 3-NH), 7.50 & 7.46 (2H, 2 × s, H-6), 6.13 & 6.12
3
(2H, 2 × t, J
6.5 Hz, H-1’), 4.45 (2H, m, H-3’), 4.26 (4H, m, H-5’),
1’, 2’
4.02 (2Н, m, H-4’), 3.39 (2H, d, 2J
10.3 Hz, CH2-P), 2.40 (4Н, m, Н-2’),
H, P
1.80 (6H, s, 5-CH3). 13С NMR, δ (DMSO-d6): 163.53 (s, C-4), 150.27 (s, C-2),
135.78 (s, C-6), 109.92 (s, C-5), 83.87 (s, C-1’), 81.43 & 81.38 (2 × d, 3J
C, P
5.6 & 4.8 Hz, C-4’), 65.89 & 65.83 (2 × d, 2J C, P 6.1 & 6.4 Hz, C-5’), 60.03 (s,
C-3’), 35.54 (s, C-2’), 12.00 & 11.92 (2 × s, 5-CH3), -14.61 (d, 1J C, P 151.0 Hz,
CH2-P). 31P NMR, δ (DMSO-d6): 15.58 (s). ESI MS, m/z (% rel. int.): 743.15
(M + Na+, 100%), 721.2 (M + H+, 48.4%).
Bis-(3’-azido-3’-deoxythymidin-5’-O-yl) azidomethylphosphonate (2f)
Obtained 75 mg (yield 59%) as a colorless viscous liquid. 1H NMR, δ
(DMSO-d6): 11.28 (2H, s, 3-NH), 7.49 & 7.46 (2H, 2 × s, H-6), 6.13 & 6.12

New Dinucleoside Phosphonate Derivatives of AZT and 3TC
77
(2H, 2 × t, 3J 1’, 2’ 6.5 Hz, H-1’), 4.43 (2H, m, H-3’), 4.29 (4H, m, H-5’), 4.00
2
(2Н, m, H-4’), 3.92 (2H, d, J
11.8 Hz, CH2-P), 2.38 (4H, m, H-2’),
CH2, P
1.79 (6H, s, 5-CH3); 13C NMR, δ (DMSO-d6): 163.56 (s, C-4), 150.27 (s, C-2),
135.85 (s, C-6), 109.90 (s, C-5), 83.88 (s, C-1’), 81.33 & 81.28 (2 × d, 3J
C, P
4.8 Hz, С-4’), 65.58 & 65.53 (2 × d, 2J C, P 5.6 Hz, С-5’), 59.94 (s, C-3’), 44.60
1
(d, J
152.6 Hz, CH2-P), 35.47 (s, C-2’), 11.86 & 11.82 (2 × s 5-CH3);
С, P
31P NMR, δ (DMSO-d6): 25.06 (s). ESI MS, m/z (% rel. int.): 659.4 (M +
Na++H+, 36.0%), 658.3 (M + Na+, 100%), 636.3 (M + H+, 48.7%)
(3’-Azido-3’-deoxythymidin-5’-O-yl) (β-L-2’,3’-dideoxy-3’-thiacytidin-5’-O-yl)
phenoxymethylphosphonate (3)
Obtained 58 mg (yield 45%) as a colorless viscous liquid. 1H NMR, δ
3
(DMSO-d6): 11.31 (1H, s, 3-NH (AZT)), 7.71 & 7.70 (1H, 2 × d, J
H-6, H-5
7.5 Hz, H-6 (3TC)), 7.51 & 7.48 (1H, 2 × q, 4J
1.1 Hz, H-6 (AZT)),
7.28 (2H, m, o-Ph), 7.20 (2H, br.s, NH2 (3TC)), 6.98 (3H, m, m-Ph & p-Ph),
H-6, 5-CH3
6.25 & 6.24 (1H, 2 × dd, 3J
5.5 Hz, 3J
5.1 Hz, H-1’ (3TC)), 6.13 (1H,
6.9 Hz, H-1’ (AZT)), 5.73 (1H,
1’, 2’a
1’, 2’b
t + dd, 3J
6.7 Hz, 3J
6.7 Hz, 3J
1’, 2’
1’, 2’a
1’, 2’b
d, 3J
7.5 Hz, H-5 (3TC)), 5.37 (1H, 2 × t, 3J
4.8 Hz, H-4’(3TC)),
4’, 5’
H-6, H-5
2
4.55 & 4.54 (2H, 2 × d, J
9.9 Hz, CH2-P), 4.46 (1H, m, H-3’ (AZT),
CH2, P
4.38 (2H, m, H-5’ (AZT)), 4.32 (2H, m, H-5’ (3TC)), 4.03 (1H, m, H-4’
(AZT)), 3.38 (1H, dd, 3J
5.3 Hz, J
11.5 Hz, H-2’a (3TC)), 3.09
2
2’a, 1’
2’a, 2’b
(1H, dd, 3J
6.6 Hz, J
11.5 Hz, H-2’b (3TC)), 2.37 (2H, m, H-2’
2
2’b, 1’
2’b, 2’a
(AZT)), 1.74 & 1.72 (3H, 2 × d, 4J 5-CH , H-6 1.1 Hz). 13С NMR, δ (DMSO-d6):
3
165.52 (s, C-4 (AZT)), 163.53 (s, C-4 (3TC)), 158.07 (d, 3J C, P 14.2 Hz, i-Ph),
154.48 (s, C-2 (AZT)), 150.30 (s, C-2 (3TC)), 140.49 (s, C-6 (3TC)), 135.72
(s, C-6 (AZT)), 129.43 (s, m-Ph), 121.55 (s, p-Ph), 114.48 & 114.44 (2 × s,
o-Ph), 109.96 & 109.92 (2 × s, C-5 (AZT)), 94.43 (s, C-5 (3TC)), 87.13 &
87.09 (2 × s, C-1’ (3TC)), 83.71 & 83.68 (2 × s, C-1’ (AZT)), 81.41 & 81.37
(2 × d, 3J
6.1 & 5.0 Hz, C-4’ (3TC)), 81.17 & 81.11 (2 × d, 3J
5.7 &
C, P
6.4 & 6.0 Hz, C-5’ (3TC)),
C, P
5.9 Hz, C-4’ (AZT)), 67.18 & 67.07 (2 × d, 2J
C, P
65.49 & 65.42 (2 × d, 2J C, P 6.7 & 5.4 Hz, C-5’ (AZT)), 61.16 & 61.02 (2 × d,
1J C, P 167.3 & 165.9 Hz, CH2-P), 60.07 (s, C-3’ (AZT)), 35.36 & 35.50 (2 × s,
C-2’ (AZT)), 35.09 & 35.07 (2 × s, C-2’ (3TC)), 11.96 & 11.93 (2 × s, 5-CH3
(AZT)). 31P NMR, δ (DMSO-d6): 26.09 (s) & 25.68 (s). ESI MS, m/z (% rel.
int.): 672.2 (M + Na++H+, 31.6%), 671.2 (M + Na+, 100.0%), 650.3 (M +
2 × H+, 27.4%), 649.2 (M + H+, 68.9%).
ABBREVIATIONS
HIV-1 human immunodeficiency virus type 1
AZT
3TC
FLT
3ꢁ-azido-3ꢁ-deoxythymidine
β-L-2ꢁ,3ꢁ-dideoxy-3ꢁ-thiacytidine
3ꢁ-fluoro-3ꢁ-deoxythymidine
PMPA 9-[2-(R)-(phosphonomethoxy)propyl]adenine

78
P. N. Solyev et al.
N ,N ꢁ-dicyclohexylcarbodiimide
DCC
TPS-Cl 2,4,6-triisopropylbenzenesulfonyl chloride.
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