New lipophilic derivatives of AZT and d4T 5′-phosphonates

Article abstract of DOI:10.1081/NCN-120022718

5′-Aminocarbonylphosphonyl and aminocarbonylmethylphosphonyl diesters of AZT and d4T were synthesized as potential anti-HIV agents.

Full text of DOI:10.1081/NCN-120022718

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New Lipophilic Derivatives of AZT and d4T 5′-
Phosphonates
Elena A. Shirokova a c , Maxim V. Jasko a , Anastasiya L. Khandazhinskaya a , Dmitry V.
Yanvarev a , Yury S. Skoblov a , Tatyana R. Pronayeva b , Nina V. Fedyuk b , Andrey G.
Pokrovsky b & Marina K. Kukhanova a
a Engelhardt Institute of Molecular Biology Russian Academy of Sciences , Moscow, Russia
b SRC Virology and Biotechnology “Vector” , Koltsovo, Russia
c Engelhardt Institute of Molecular Biology , 32, Vavilov Str., Moscow, 119991, Russia
Published online: 31 Aug 2006.
To cite this article: Elena A. Shirokova , Maxim V. Jasko , Anastasiya L. Khandazhinskaya , Dmitry V. Yanvarev , Yury S.
Skoblov , Tatyana R. Pronayeva , Nina V. Fedyuk , Andrey G. Pokrovsky & Marina K. Kukhanova (2003) New Lipophilic
Derivatives of AZT and d4T 5′-Phosphonates, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 981-985, DOI: 10.1081/
NCN-120022718
To link to this article: http://dx.doi.org/10.1081/NCN-120022718
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 981–985, 2003
New Lipophilic Derivatives of AZT and d4T 50-Phosphonates
1
Elena A. Shirokova,1, Maxim V. Jasko,
*
Anastasiya L. Khandazhinskaya,1 Dmitry V. Yanvarev,1
Yury S. Skoblov,1 Tatyana R. Pronayeva,2 Nina V. Fedyuk,2
Andrey G. Pokrovsky,2 and Marina K. Kukhanova1
1Engelhardt Institute of Molecular Biology Russian Academy of Sciences,
Moscow, Russia
2SRC Virology and Biotechnology ‘‘Vector’’, Koltsovo, Russia
ABSTRACT
50-Aminocarbonylphosphonyl and aminocarbonylmethylphosphonyl diesters of
AZT and d4T were synthesized as potential anti-HIV agents.
Key Words: Anti-HIV agents; AZT and d4T derivatives; Nucleoside 50-phos-
phonates.
Going on the studies of antiviral agents in the series of nucleotide derivatives, we
paid attention on AZT conjugated with phosphonoformic (PFA) and phosphono-
acetic (PAA) acids. Earlier diesters and triesters of type (I) were prepared.[1–3] Their
antiviral effect was much higher than that of PFA and comparable with that of AZT.
A poor increase in the efficacy could be due to high instability in water solutions of
the triesters and poor penetration through cell membranes of the diesters.[3]
*Correspondence: Elena A. Shirokova, Engelhardt Institute of Molecular Biology, 32, Vavilov
Str., Moscow 119991, Russia; Fax: +7 095 135 1405; E-mail: aak@imb.ac.ru.
981
DOI: 10.1081/NCN-120022718
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

982
Shirokova et al.
We synthesized a set of uncharged 50-aminocarbonyl- and 50-aminocarbonyl-
methylphosphonate derivatives of AZT and d4T (II–V) and evaluated their activity
in cell cultures infected with HIV-1.
AZT-derived
R0
d4T-derived
R0
Comp
n
R
Comp
n
R
IIa
IIb
IIc
0
0
0
H
Me
PhCH2CH2
Et
Et
Et
IVa
IVb
IVc
0
0
0
H
Me
PhCH2CH2
Et
Et
Et
IIIa
IIIb
IIIc
1
1
1
H
H
Me
Et
cyclohexyl
Et
Va
–
–
1
H
Et
The target amides II–V were prepared by treatment of the corresponding 50-
ethoxycarbonylphosphonyl nucleosides with primary amines followed by esterifica-
tion with various alcohols. Esterification was carried out using two procedures: in
the presence of TPSCl and under Mitsunobu conditions. In the case of secondary
alcohols, the first method was preferential, whereas for primary alcohols the second
procedure resulted in higher yields.
The resulting compounds were mixtures of diastereoisomers (due to a chiral
phosphorus atom). Isomers A and B of amide IIa were separated by reverse-phase
chromatography, their retention times differing in about 2 min.
Chemical stability of the synthesized amides was estimated in 0.05 M phosphate
buffer at 37ꢀC. Figure 1 shows the stability of amide IIa under various pH.
Obviously, it was strongly pH-dependent. In the range of pH 5.5–7.8 its half-life time
decreased from 6 h to less than 30 min. At physiological pH 7.2–7.4 its half-life time
was about 1 h.
The composition of the reaction mixture in the process of chemical hydrolysis
of amide IIa was studied at pH 7.2 at 37ꢀC. We observed the formation of two
products, a major one being AZT and a minor one - 50-aminocarbonylphospho-
nyl-AZT. The formation rate of the former was considerably higher than that of
the latter (Fig. 2). The corresponding PAA-AZT analogue was degraded under simi-
lar conditions to give only the nucleoside.
As a whole, PAA derivatives in both series were noticeably more stable than the
corresponding PFA ones (for example, 7 h for IVa vs. ꢁ 24 h for V). d4T derivatives

New Lipophilic Derivatives of AZT
983
Figure 1. pH-Dependence of half-life time of amide IIa.
had longer half-lives than the corresponding AZT derivatives (7 h for IVa against
2.5 h for IIa). The substitution at the amide group increased the chemical stability
(2.5 h for IIa, 6 h for IIb and > 10 h for IIc).
Antiviral effect of the compounds under study was evaluated in MT-4 cells
infected with HIV-1 strain GKV-4046 (Table 1). The tested compounds were added
into the culture after the virus adsorption. The activity was measured by the amount
of viral p24 antigen using immunoassay. Cytotoxicity was measured by the colori-
metric method.
Figure 2. Chemical hydrolysis of amide IIa in phosphate buffer at pH 7.2.

984
Shirokova et al.
Table 1. Anti-HIV properties of the synthesized compounds.
ꢂ
ꢂꢂ
ꢂꢂ
CD50
(mM)
ID50
(mM)
ID90
(mM)
ISꢂꢂꢂ
IIa
R ¼ Et, R1 ¼ H
IIb
298.3
0.0009
0.004
0.007
0.0002
346800
112.9
177.7
0.00024
470400
R ¼ Et, R1 ¼ Me
IIc
R ¼ Et,
< 0.0002
> 888500
R1 ¼ PhCH2CH2
IIIa
R ¼ Et, R1 ¼ H
IIIc
456.3
170.1
0.29
1.68
–
1573
0.0085
20012
R ¼ cyclohexyl,
R1 ¼ H
IVa
573.5
85.7
0.9
2.1
637
R ¼ Et, R1 ¼ H
IVb
0.04
0.27
2187
R ¼ Et, R1 ¼ Me
V
267.9
5.4
18.8
50
R ¼ Et, R1 ¼ H
AZT
d4T
187.5
314
0.018
0.24
0.35
23.8
11030
1308
ꢂCD50 – Compound concentration required for 50% inhibition of cell proliferation.
ꢂꢂID50 and ID90 – Compound concentration required for 50% or 90% inhibition of HIV repro-
duction, respectively.
ꢂꢂꢂSI – selectivity index, CD50=ID50.
As is seen from the table, this modification seems to be promising in the case of
PFA-AZT series: as a rule a significant increase in the activity resulted in an increase
of the selectivity index. For d4T analogues, this gain was not so obvious.
ACKNOWLEDGMENTS
The work was supported by ISTC, project 1244, and Russian Foundation for
Basic Research, project 02-04-49009.
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human immunodeficiency virus type 1 replicatıon by phosphonoformate esters
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288–294.

New Lipophilic Derivatives of AZT
985
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