Phosphorodiamides as prodrugs for antiviral nucleosides

Article abstract of DOI:10.1081/NCN-120022696

New phosphorodiamides of modified nucleoside monophosphates were synthesized and their antiviral properties were evaluated.

Full text of DOI:10.1081/NCN-120022696

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Phosphorodiamides as Prodrugs for Antiviral
Nucleosides
Alexander V. Shipitsyn a c , Natalya F. Zakirova a , Evgeny F. Belanov b , Tatyana R. Pronyaeva
b , Nina V. Fedyuk b , Marina K. Kukhanova a & Andrey G. Pokrovsky b
a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , Moscow, Russia
b SRC Virology and Biotechnology “Vector” , Koltsovo, Russia
c Englelhardt Institute of Molecular Biology , 32, Vavilov Str., Moscow, 119991, Russia
Published online: 31 Aug 2006.
To cite this article: Alexander V. Shipitsyn , Natalya F. Zakirova , Evgeny F. Belanov , Tatyana R. Pronyaeva , Nina V. Fedyuk ,
Marina K. Kukhanova & Andrey G. Pokrovsky (2003) Phosphorodiamides as Prodrugs for Antiviral Nucleosides, Nucleosides,
Nucleotides and Nucleic Acids, 22:5-8, 963-966, DOI: 10.1081/NCN-120022696
To link to this article: http://dx.doi.org/10.1081/NCN-120022696
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 963–966, 2003
Phosphorodiamides as Prodrugs for Antiviral Nucleosides
1
2
Alexander V. Shipitsyn,1, Natalya F. Zakirova, Evgeny F. Belanov,
Tatyana R. Pronyaeva,2 Nina V. Fedyuk,2 Marina K. Kukhanova,1
and Andrey G. Pokrovsky2
*
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,
Moscow, Russia
2SRC Virology and Biotechnology ‘‘Vector’’, Koltsovo, Russia
ABSTRACT
New phosphorodiamides of modified nucleoside monophosphates were synthe-
sized and their antiviral properties were evaluated.
INTRODUCTION
Nucleoside monophosphate esters and their analogues displaying activity
towards a wide range of viruses as well as the corresponding phosphoroamide deri-
vatives have been thoroughly studied.[1,2] The mechanism of action of these nucleo-
sides involves a cascade of intracellular triphosphorylation followed by inhibition of
viral polymerases by the resulting triphosphates. The use of monophosphate deriva-
tives allows bypass of the first stage of triphosphorylation cascade, which is often
a limiting stage.
*Correspondence: Alexander V. Shipitsyn, Englelhardt Institute of Molecular Biology, 32,
Vavilov Str., Moscow 119991, Russia; Fax: þ7 095 135 1405; E-mail: aal@imb.ac.ru.
963
DOI: 10.1081/NCN-120022696
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Shipitsyn et al.
Scheme 1.
Nucleoside phosphorodiamidates are studied to a lesser extent. However, for
some members of this class, a decrease in toxicity was observed[3,4]. The goal of this
work was the synthesis of new nucleoside 50-phosphorodiamidates and evaluation of
their antiviral properties.
RESULTS AND DISCUSSION
Dimorpholidates (Ia–f) were prepared by the reaction of the corresponding
nucleoside with phosphorus oxychloride followed by treatment of the resulting phos-
phodichloridate with an excess of morpholine in methanol (Sch. 1).
In the case of AZT and d4T the yield was about 60%.
Bis(methoxyamides) (IIIa,b) and bis(diphenylmethylamides) (IVa,b) exemplify
another type of AZT and d4T monophosphate derivatives. They were synthesized
analogously to dimorpholidates (Ia,b) in yields of 45 and 35% and 30 and 25%,
respectively. For the synthesis of diamides (III) we used methoxyamine hydrochlor-
ide and an excess of triethylamine to remove the releasing hydrogen chloride. In the
case of diamides (IV) dioxane was taken as a solvent, since both diphenylmethyla-
mine and methanol reacted with the intermedia phosphodichloridate to give a
considerable amount of phosphoromethoxyamidate.

Phosphorodiamides as Prodrugs
965
Table 1.
Compound CD50 (mM)a ID50 mM)b SIc Compound CD50 (mM)a ID50 (mM)b
SIc
AZT
Ia
IIIa
IVa
187.5
>206
740
0.018
7.2
0.012
1.7
10,400
>30
61,700
3.6
d4T
Ib
IIIb
IVb
314
>226
828
0.01
31,400
0.0022
0.028
0.55
>1,00,000
29,600
77
6.2
47.3
aCD50 – 50% cytotoxic dose.
bID50 – 50% inhibiting dose.
cSI – selectivity index (CD50=ID50 ratio).
The results of biochemical tests of these compounds are shown in Table 1. The
amides were evaluated in MT-4 cells infected with HIV-1 strain GKV-4046. As is
seen from the table, diamides (I) and (III) of the AZT and d4T series dramatically
differed in their antiviral activity. For example, dimorpholidate Ib was substantially
more active than the parent nucleoside, whereas in the case of compounds (III), this
modification was more advantageous for the AZT series. It is interesting that the
activity of monomorpholidate AZT (IIa) prepared according to Sch. 1 with an addi-
tional stage of treating the intermediate phosphorodichloridate with water, displayed
the activity similar to that of Ib (ID50 0.0023 mM and SI ꢀ 1,00,000). High toxicity of
AZT bis(diphenylmethylamido)phosphate is also noteworthy.
Phosphorodimorpholidates of d4T (Ic) and acyclovir (Id) were synthesized
according to Sch. 1 in yields of 70 and 30%, respectively. Both of them were inactive
against HIV-1 and HSV-1, respectively.
Using this scheme, we also prepared phosphorodimorpholidate of arabinoade-
nosine (Ie), which is active against many viruses but is highly toxic.[5] Unlike thymi-
dine derivatives, we obtained a mixture of phosphoromono- and -dimorpholidates
(IIe, Ie) (15 and 55%, respectively). The tests with cell cultures infected with cytome-
galovirus and poxviruses are in progress.
Adenosine N1-oxide is known to be rather active against the poxvirus group.[5]
We recently showed that it was highly active against smallpox virus with ID50
0.57 mM, the CD50 being 19 mM. With the goal to decrease the toxicity, we synthe-
sized its phosphorodimorpholidate (Ig).
However, direct interaction of the nucleoside with POCl3 and morpholine under
aforementioned conditions yielded less than 10% of the target (Ig). As an alternative,

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Shipitsyn et al.
we synthesized compound (If) and oxidized it with m-chloroperbenzoic acid in
methanol in the presence of tributylamine (Sch. 2). The overall yield of phosphoro-
dimorpholidate (Ig) was 35%.
It is noteworthy that while preparing riboadenosine derivative (If), we isolated
about 10% of monomorpholidate (IIf) in addition to the target diamide.
Thus, the introduction of a phosphorodiamidate residue in the molecule of
nucleosides displaying antiviral activity in some cases strongly affects their antiviral
properties. Therefore, further investigations in this field seem to be promising.
ACKNOWLEDGMENTS
The work was supported by International Science and Technology Center (ISTC
projects 1244 and 1989) and RF Ministry of Industry, Sciences and Technology, pro-
ject 31.
REFERENCES
1. Perigaud, C.; Girardet, J.-L.; Gosselin, G.; Imbach, J.-L. Advances in Antiviral
Drug Design 1996, 2, 147–172.
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3. Phelps, M.E.; Woodman, P.W.; Danenberg, P.V. J. Med. Chem. 1980, 23, 1232–
1235.
4. Starrett, J.E., Jr.; Tortolani, D.R.; Russell, J.; Hitchcock, M.J.M.; Whiterock,
V.; Martin, J.C.; Mansuri, M.M. J. Med. Chem. 1994, 37, 1857–1864.
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