Synthesis and antiherpetic activity of acyclovir phosphonates

Article abstract of DOI:10.1081/NCN-120021431

Phosphonate derivatives of acyclovir containing phosphorous acid and ethoxy-carbonylphosphonic acid residues as well as their isopropyl esters were prepared. They selectively inhibited the herpes simplex virus 1 reproduction in Vero cell culture, the effi

Full text of DOI:10.1081/NCN-120021431

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Synthesis and Antiherpetic Activity of Acyclovir
Phosphonates
Inna L. Karpenko a , Maxim V. Jasko a , Valeriya L. Andronova b , Alexander V. Ivanov a
Marina K. Kukhanova a , Georgy A. Galegov b & Yury S. Skoblov a c
,
a Engelhardt Institute of Molecular Biology, Russian Academy of Medical Sciences, Moscow,
Russian Federation
b Ivanovskii Institute of Virology, Russian Academy of Medical Sciences, Moscow, Russian
Federation
c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov str.,
Moscow, 119991, Russian Federation
Published online: 07 Feb 2007.
To cite this article: Inna L. Karpenko , Maxim V. Jasko , Valeriya L. Andronova , Alexander V. Ivanov , Marina K. Kukhanova ,
Georgy A. Galegov & Yury S. Skoblov (2003): Synthesis and Antiherpetic Activity of Acyclovir Phosphonates, Nucleosides,
Nucleotides and Nucleic Acids, 22:3, 319-328
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, No. 3, pp. 319–328, 2003
Synthesis and Antiherpetic Activity of
Acyclovir Phosphonates
Inna L. Karpenko,1 Maxim V. Jasko,1 Valeriya L. Andronova,2
Alexander V. Ivanov,1 Marina K. Kukhanova,1
Georgy A. Galegov,2 and Yury S. Skoblov1,
*
1Engelhardt Institute of Molecular Biology and 2Ivanovskii Institute of Virology,
Russian Academy of Medical Sciences, Moscow,
Russian Federation
ABSTRACT
Phosphonate derivatives of acyclovir containing phosphorous acid and ethoxy-
carbonylphosphonic acid residues as well as their isopropyl esters were prepared.
They selectively inhibited the herpes simplex virus 1 reproduction in Vero cell
culture, the efficacy of esters being 3-4 times higher than that of ACV. The hydro-
lysis of the synthesized compounds was studied in the PBS buffer and human
blood serum.
Key Words: Acyclovir; Herpes simplex virus type 1; Phosphonate.
INTRODUCTION
Acyclovir (9-[(2-hydroxyethyloxy)methyl]guanine) (ACV) is one of the widely
used drugs for treatment of herpes infection. The mechanism of its action involves
*Correspondence: Yury S. Skoblov, Engelhardt Institute of Molecular Biology, Russian
Academy of Sciences, 32 Vavilov str., Moscow 119991, Russian Federation; E-mail: Chizhik@
ok.ru.
319
DOI: 10.1081/NCN-120021431
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

320
Karpenko et al.
a series of successive stages, namely, phosphorylation catalyzed by viral and cellular
kinases, the HSV DNA polymerase-catalyzed incorporation of the resulting triphos-
phate into the growing DNA chain, and termination of the proviral DNA synthesis.
However, the bioavailability of acyclovir is low. Many attempts were made to
increase it by the introduction of chemical groups facilitating the cell penetration,
affecting the metabolism pathway and rate, etc.[1] This approach was successfully
realized in antiherpetic drugs ValaciclovirÕ and FamciclovirÕ.
In the case of nucleotide analogues displaying antiviral activity this ‘‘pronucleo-
tide’’ approach was also rather promising. For example, when 30-azido-30-deoxy-
thymidine and 20,30-didehydro-20,30-dideoxythymidine were modified with phosphonic
acid derivatives, the resulting phosphonates occasionally demonstrated an enhanced
anti-HIV activity and lower toxicity than those of parent nucleosides.[2,3] Moreover,
we showed earlier that acyclovir hydrogenphosphonate I was active in Vero cell
cultures infected with several HSV-1 strains including the ACV-resistant strain.[4]
It is worth noting that the modification of this type was not always successful. In
the case of 5-substituted 20-deoxyuridines, the phosphonates of this type did not offer
advantages over the parent nucleosides in respect to antiviral properties.[5–7]
The aim of our work was the synthesis of ACV ethoxycarbonylphosphonate II,
isopropyl esters III and IV, and carboxyphosphonate V and evaluation of their pro-
perties towards various HSV-1 strains including ACV-resistant ones.
RESULTS AND DISCUSSION
Several methods of synthesis of nucleoside 50-phosphonates have been described.
Ethoxycarbonylphosphonic acid was coupled with guanine-containing nucleosides in
the presence of 1,3-dicyclohexylcarbodiimide; the yields of the products did not
thereby exceed 50%.[8] To phosphonylate protected guanosine nucleosides, ethoxy-
carbonylphosphonic dichloride was used.[9] Although the yields of target products
were 72–85%, the extra stage of the preparation of phosphonylating agent can be
referred to disadvantages of the method. In the reaction of ethoxycarbonylmethyl-
phosphonic acid with ACV in the presence of TPSCl, the acid residue was added
both to the side chain and the guanine base. When N-acetyl derivative of ACV
was used in this reaction, the target product was obtained, but its yield achieved
only 9%.[6]
We used the coupling of ACV with ethoxycarbonylphosphonic acid (prepared
in situ by the treatment of triethyl phosphonoformate with Me3SiBr) in the presence
of PivCl as a condensing agent (Sch. 1). The yield of isolated phosphonate II was
74%. Isopropyl esters III IV were prepared by the coupling of isopropanol with
phosphite I in the presence of PivCl or with ethoxycarbonylphosphonate II in the
presence of TPSCl. Carboxyphosphonate V was obtained by treatment of phospho-
nate II with aqueous alkali.
The homogeneity of the compounds was controlled by HPLC; their structure
1
was confirmed by H and 31P NMR spectral data. The 31P NMR spectrum of ester
III in CD3OD represented a resonance group interpreted as ddt with the coupling
1
3
constants JP,H ¼ 712 Hz and JP,H ꢀ 9 Hz (see Fig. 1A); it was transformed into a

Acyclovir Phosphonates
321
Scheme 1.
broad singlet when the proton-phosphorus interaction was decoupled (Fig. 1B). It
is worth noting that in the case of D2O as a solvent, the hydrogen atom in the P-H
bond was substituted by a deuterium one. It was clearly observed in the pattern
of the proton-decoupled 31P-NMR spectrum, in which a singlet resonance at
1
8.6 ppm disappeared and a triplet at 8.3 ppm with JP,D ¼ 109 Hz was observed
Figure 1. 31P-NMR patterns of ester III.

322
Karpenko et al.
(Fig. 1C). When the spectrum of ester III was registered in DMSO-d6, the coupling
1
constant JP,H was decreased to the value of 698 Hz.
When planning the synthesis of hydrogenphosphonate and phosphonoformate
ACV derivatives II–V, we presumed that these compounds are actual ACV pre-
cursors. When penetrated into the cell, they will be converted in ACV with different
efficacy. Scheme 2 demonstrates possible hydrolysis pathways for the synthesized
compounds independently of the reaction mechanism.
We studied the hydrolysis of compounds I–V in PBS and in human blood serum
(Table 1); the aliquots were analyzed by HPLC analysis. Anionic phosphonates I, II
and V displayed high stability both in PBS and in human blood serum. Carboxy-
phosphonate V was extraordinarily stable: only ACV was found after 3 days of
incubation in trace quantities.
It is interesting to note that phosphonate II was hydrolyzed by two alternative
ways to give ACV or carboxyphosphonate V. The hydrolysis in PBS gave ACV
and V in a ratio of about 1 : 1, whereas carboxyphosphonate V prevailed in human
blood serum. When comparing the ACV accumulation rates from II in the aforemen-
tioned media, one can imply that carboxyphosphonate V is mainly resulted from
enzymatic hydrolysis in human blood serum, whereas ACV is a product of chemical
hydrolysis.
Chemical transformations of phosphonates IV and V could involve the forma-
tion of the corresponding hydrogenphosphonates III and I as was observed for some
phosphonoformates.[10] However, according to the data presented in Table 1, the
products of such transformation were not found.
The table demonstrates the averaged data of three independent series; the experi-
mental error for the compounds I–IV did not exceed 10%.
Scheme 2.

Acyclovir Phosphonates
323
Table 1. Stability of compounds I–V in PBS and human blood serum.
PBS
Human blood serum
Incubation
time
Relative amount of the
componentsꢁ
Incubation
time
Relative amount of the
componentsꢁ
Comp.
I
72 h
72 h
90 min
45 min
72 h
I:ACV
98:2
84:7:9
53:47
54:36:10
ꢀ100ꢁꢁ
72 h
72 h
35 min
10 min
72 h
I:ACV
82:18
38:8:54
48:52
35:47:18
ꢀ100ꢁꢁ
II
III
IV
V
II:ACV:V
III:ACV
IV:ACV:II
V
II:ACV:V
III:ACV
IV:ACV:II
V
ꢁThe amount of starting compound at t ¼ 0 min is taken as 100%.
ꢁꢁTraces of ACV were observed.
The stability of esters III and IV is substantially lower both in the buffer and in
the blood serum if compared with phosphonates I and II, respectively. It is essential
that ACV was the only detected hydrolysis product of ester III in all the conditions
under study. Since phosphonate I is hydrolytically stable, we can imply that ester III
is hydrolyzed directly to ACV (Sch. 2).
ACV and phosphonate II were identified as hydrolysis products of ester IV both
in the buffer and blood serum. Ester III was not found. The major product was also
ACV, although the rate of accumulation of phosphonate II was higher in the blood
serum (Fig. 2).
We studied antiherpetic activity of the synthesized phosphonates I–V in Vero
cell culture infected with various HSV-1 strains. According to the data presented
in Table 2, compounds I–V are low toxic. They completely inhibited virus reproduc-
tion (ID95) in Vero cells infected with HSV-1=L2 at nontoxic concentrations.
Uncharged esters III and IV were more effective than ACV. Compounds II–V
were inactive towards laboratory strain of ACV-resistant herpes simplex virus
Figure 2. Hydrolysis of ester IV in PBS (A) and human blood serum (B).

324
Karpenko et al.
Table 2. Inhibition of HSV-1 reproduction in Vero cell culture with compounds I–V.
HSV-1=L2
HSV-1=L2=R
ID50 ID95
HSV-1 (Avd)
ID50 ID95
CTD50 ID50
mM
ID95
mM
Comp.
mM
SI
mM
mM
SI
96
mM
mM
SI
I
3922
20.4
> 2646 330.7 661
81.7
192
> 8
40.8
331
327
661 > 8
20.4
–
81.7
–
192
–
II
III
IV
V
> 3021
> 2481
> 2857
> 2226
0.45
0.37
35.7
1.82
1.89 > 6713 181
0.77 > 6705 149
> 363 > 16.7
> 298 > 16.7
> 286 > 10
> 486 > 4.6
1.9
3.8 > 1590
3.1 > 3222
0.77
143
6.3 101
71.4
> 80
2.27 > 1223 486
286
286
> 20
353
ACV
CTD50 – 50% cytotoxic dose for uninfected Vero cells; ID50 and ID95 – doses inhibiting the
viral cytopathogenic effect by 50 and 95%, respectively; SI – selectivity index (CTD50=ID50
ratio).
(HSV-1=L2=R). This fact indirectly supports our hypothesis about the transforma-
tion of compounds II–V into ACV. At the same time esters III IV were substantially
active towards the clinical isolate of HSV-1 (Avd) strain weakly resistant to ACV.
Their potency may be accounted for by more effective penetration through cell
membranes and, as a consequence, by higher intracellular ACV concentration. More
thorough studies of cellular metabolism of compounds III and IV will allow the
evaluation of the correctness of this assumption.
The analysis of the data on hydrolysis of compounds I–V in PBS and blood
serum shows that only some of the possible transformations presented on Scheme
2 are realized. If to compare these data with the ones on antiherpetic activity, one
can assume that the rate and mechanism of hydrolysis of compounds I and V in Vero
cells differed from those in blood serum. This hypothesis is based on the observed
discrepancy between high hydrolytic stability and pronounced antiviral activity of
phosphonates I and V (Tables 1 and 2).
EXPERIMENTAL
Triethyl phosphonoformate, PivCl, and Dowex 50 W X 8 were from Fluka,
TPSCl was from Aldrich. Hydrogenphosphonate I was obtained as described in
Ref.[4].
UV spectra were recorded on a Shimadzu UV-2401PC spectrophotometer
1
(Japan). H NMR spectra (400 MHz) and 31P NMR spectra (162 MHz) were regis-
tered on a Bruker AMX III-400 spectrometer (Germany). Column chromatography
was performed on DEAE-Toyopearl 650 M (Toyo Soda, Japan), Kieselgel 60
(63–100 mm), LiChroprep RP-8, and RP-18 (25–40 mm) (Merck, Germany). HPLC
was performed on a Gilson chromatograph (France) using a Lichrosorb RP-18
(7 mm, Merck) column (4 ꢂ 150 mm) in a gradient of MeOH in 5 mM sodium phos-
phate buffer (pH 4.8); gradient A: 0% for 5 min, 0 ! 7% for 5 min, 7 ! 17% for
25 min, and 17 ! 66% for 5 min; gradient B: 0% for 5 min; 0 ! 7% for 5 min,

Acyclovir Phosphonates
325
7 ! 17% for 25 min, 17 ! 53% for 5 min, 53 ! 66% for 5 min, and 66% for 5 min;
flow rate 0.4 mL=min; detection at 254 nm.
Acyclovir Ethoxycarbonylphosphonate, Ammonium Salt (II). Trimethylbromo-
silane (1.8 mL, 13.9 mmol) was dropped to a precooled (0ꢃC) solution of triethyl
phosphonophormate (1 mL, 5.28 mmol) in carbon tetrachloride (3 mL), and after
18 h at 20ꢃC the solution was evaporated and coevaporated with toluene
(3 ꢂ 5 mL). The residue was dissolved in 2 mL of pyridine and 2 mL of water and
after 1 h the solution was evaporated and coevaporated with pyridine (3 ꢂ 5 mL).
The residue was dissolved in pyridine (3 mL) and added to the suspension of acyclo-
vir (770 mg, 3.4 mmol) in DMF (3 mL). The mixture was cooled (0ꢃC) and PivCl
(1 mL, 8.1 mmol) was added. After stirring at 5ꢃC for 18 h, the reaction mixture
was diluted with water (50 mL), and the solution was loaded on a DEAE-Toyopearl
column (3 ꢂ 10 cm). The product was eluted in a linear gradient of NH4HCO3
(0 ! 0.1 M, 1 l). The target fractions were concentrated and repurified on a LiChro-
prep RP-8 column (3 ꢂ 25 cm) by eluting with water. The target fractions were
freeze-dried to give 948 mg (74%) of compound II; retention time 26 min (gradient
1
A). UV (H2O, pH 7): lmax 254 nm. H-NMR (CD3OD, d, ppm, J, Hz): 7.88s (1H,
H-8), 5.50s (2H, CH2Gua), 4.18q (2H, JCH ,CH 7.2, CH2-Et), 4.11m (2H, CH2OP),
2
3
3.75dd (2H, J 4.4, 5, CH2OC), 1.28t (3H, CH3-Et). 31P-NMR (CD3OD, d, ppm, J,
Hz): ꢄ4.51t (J 7).
Acyclovir P-(isopropyl) Hydrogenphosphonate (III). To a precooled (0ꢃC) solu-
tion of hydrogenphosphonat I (71 mg, 0.23 mmol) and isopropanol (0.5 mL) in pyri-
dine (5 mL), PivCl (100 mL, 0.8 mmol) was added, and the mixture was kept
overnight at 5ꢃC. After dilution with water (10 mL), the reaction mixture was evapo-
rated in a vacuum, coevaporated with water (3 ꢂ 5 mL), and the residue was purified
on a LiChroprep RP-8 column (2 ꢂ 18 cm) eluting in a linear gradient of MeOH
(0 ! 20%, 1 l) in water. The target fractions were evaporated, dissolved in water
and freeze-dried to yield 43 mg (56%) of compound III; retention time 42 min (gra-
1
dient B). UV (MeOH): lmax 254 nm. H-NMR (DMSO-d6): 10.62s (1H, NH), 7.82s
(1H, H-8), 6.79d (1H, JH,P 698, H-P), 6.51s (2H, NH2), 5.37s (2H, CH2Gua), 4.55m
(1H, CH-iPr), 4.05m (2H, CH2OP), 3.66t (2H, J 4.4, CH2OC), 1.23d (6H, JCH ,CH
3
3
3
6.2, CH3-iPr). 31P-NMR (DMSO-d6): 8.16ddt (1JH,P 698, JP,CH ꢀ JP,CH 9.2).
2
Acyclovir P-(isopropyl) Ethoxycarbonylphosphonate (IV). To a precooled (5ꢃC)
solution of phosphonate II (76 mg, 0.2 mmol) in pyridine (5 mL) and isopropanol
(0.5 mL), TPSCl (240 mg, 0.8 mmol) was added in two portions during 3 h, the reac-
tion mixture was kept overnight at 5ꢃC, evaporated in a vacuum, coevaporated
with water (3 ꢂ 5 mL) and the residue was purified by column chromatography on
LiChroprep RP-8 (2 ꢂ 18 cm) eluting in a linear gradient of MeOH (0 ! 30%, 0.5 L)
in water. The fractions containing compound IV were evaporated, coevaporated with
EtOH, dissolved in CHCl3 (1.9 mL) and MeOH (0.1 mL) and chromatographed on
a silica gel column (2.5 ꢂ 25 cm) in a mixture of CHCl3-MeOH 9 : 1 (v=v). The
target fractions were evaporated, the residue was dissolved in water and freeze-dried
to give 31 mg (38%) of compound IV; retention time 43 min (gradient B). UV (MeOH):
lmax 254 nm. 1H-NMR (CD3OD): 7.87s (1H, H-8), 5.49s (2H, CH2Gua),

326
Karpenko et al.
4.79m (1H, CH-iPr), 4.31m (4H, CH2OP, CH2-Et), 3.83dd (2H, J 3.7, 4.7, CH2OC),
1.30–1.36m (9H, CH3-iPr, CH3-Et). 31P-NMR (CD3OD): ꢄ4.87dt (3JP,CH ꢀ JP,CH 7.1).
3
2
Acyclovir Carboxyphosphonate, Ammonium Salt (V). A solution of 1 M sodium
hydroxide (600 mL, 0.6 mmol) was dropped to a precooled (5ꢃC) solution of phos-
phonate II (70 mg, 0.19 mmol) in water (5 mL). After stirring at 20ꢃC for 18 h the
reaction mixture was loaded on a Dowex-50 (NH4þ-form) column (3.5 ꢂ 10 cm)
and eluted with water. The eluate was evaporated, and the residue was purified on
a LiChroprep RP-18 column (2 ꢂ 18 cm) eluting with water. The target fractions were
freeze-dried to give 46 mg (74%) of compound V; retention time 11 min (gra-
1
dient A). UV (H2O, pH 7) : lmax 254 nm. H-NMR (D2O): 7.95s (1H, H-8), 5.53s
(2H, CH2Gua), 4.03m (2H, CH2OP), 3.78m (2H, CH2OC). 31P-NMR (D2O): 1.48br.s.
Experiments with Cell Cultures
Vero cells culture (African green monkey kidney cells) was from the Laboratory
of Tissue Culture, Institute of Virology. The cells were maintained on Eagle’s
medium supplemented with 5% fetal calf serum (Fluka).
HSV-1=L2 strain from the virus collection of the Institute of Virology was used.
The mutant strain HSV-1=L2=R resistant to acyclovir was obtained by serial
passages in the presence of increasing concentrations of ACV at low multiplicity
of infection (0.01 PFU=cell). The resulting viral strain was used to obtain the viral
clone resistant to ACV (for the protocols, see Ref.[11]). Clinical isolate of acyclovir-
resistant HSV-1 strain was obtained by infecting Vero cells with virus – containing
material that was taken from patients according to Ref.[12]. Viral strains were passed
in a 1:1 mixture of Eagle’s and 199 media supplemented with 2% fetal calf serum.
For cell cultivation and infection, 96-well plastic plates (Linbro, Flow Lab., UK)
were used.
The antiviral activity was measured by the capacity of the compounds under
study to inhibit the development of virus-induced cytopathogenic effect (CPE) by
50% (ID50) and 95% (ID95) as compared with CPE in the control infected cultures.[13]
The multiplicity of infection was 0.1 PFU=cell. The infection titer was determined
after 48-h incubation of the infected culture.
Cytotoxicity was measured by the capacity of dead cells to be stained with
Trypan Blue.[14] The CTD50 value was determined as the concentration of the compound
that provided 50% viability of the cells after 72-h contact with the compound under study.
Hydrolysis of Compounds in PBS and Human Serum
Hydrolysis of compounds was studied in the PBS (pH 7.2) (0.14 M NaCl,
1.5 mM KH2PO4, 2.7 mM KCl, and 8.1 mM Na2HPO4). The compound under study
(5 mL, 10 mM aqueous solution) was added to the PBS (95 mL) and incubated at
37ꢃC. After a certain interval (Table 1) an aliquot (15 mL) was concentrated on an
Automatic Speed-Vac Concentrator AS260 (USA). The residue was diluted with
5 mM sodium phosphate buffer (10 mL, pH 4.8) and analyzed by HPLC. For
compounds I, II, V gradient A and for esters III IV gradient B were used.

Acyclovir Phosphonates
327
Human blood serum (95 mL) was added to 10 mL aqueous solution of the tested
compound (5 mL), and the mixture was incubated at 37ꢃC. An aliquot (15 mL) was
taken, MeOH (45 mL) was added, and the mixture was kept for 20 min at ꢄ20ꢃC.
The mixture was centrifuged on an Eppendorf Centrifuge 5415 (Germany) at
15,800 g, the supernatant was concentrated on an Automatic Speed-Vac Concentra-
tor AS260. The sample was diluted with 5 mM sodium phosphate buffer (10 mL,
pH 4.8) and analyzed by HPLC.
ABBREVIATIONS
ACV
HSV-1=L2
PivCl
acyclovir
herpes simplex virus type 1
pivaloyl chloride
TPSCl
PBS
2,4,6-triisopropylbenzenesulfonyl chloride
the phosphate buffer solution
ACKNOWLEDGMENT
The work was supported by the Russian Foundation for Basic Research,
projects No. 01-04-48627, No. 02-04-49009, and No. 02-04-06525.
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Received August 14, 2002
Accepted January 27, 2003