Design and synthesis of vidarabine prodrugs as antiviral agents

Article abstract of DOI:10.1016/j.bmcl.2008.12.031

5′-O-d- and l-amino acid derivatives and 5′-O-(d- and l-amino acid methyl ester phosphoramidate) derivatives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi- or more potent in vitro than vidarabine against two pox v

Full text of DOI:10.1016/j.bmcl.2008.12.031

Bioorganic & Medicinal Chemistry Letters 19 (2009) 792–796
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of vidarabine prodrugs as antiviral agents
a
a
a
a
b
Wei Shen a, , Jae-Seung Kim , Phillip E. Kish , Jie Zhang , Stefanie Mitchell , Brian G. Gentry ,
*
Julie M. Breitenbach b, John C. Drach b, John Hilfinger a
a TSRL Inc., 540 Avis Drive Suite A, Ann Arbor, MI 48108, USA
b Department of Biologic & Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
50-O- -amino acid derivatives and 50-O-(
D- and L D- and L-amino acid methyl ester phosphoramidate) deriv-
Article history:
Received 17 September 2008
Revised 3 December 2008
Accepted 4 December 2008
Available online 10 December 2008
atives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi- or
more potent in vitro than vidarabine against two pox viruses and their uptake by cultured cells was
improved compared to the parent drug.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Vidarabine
ara-A
Amino acid ester prodrug
Phosphoramidate prodrug
Antiviral activity
Selective protection of nucleoside
Levulinate ester
Oral bioavailability
Adenosine deaminase
Arahypoxanthine
ara-H
Caco-2 permeability
There has been continued interest in the synthesis and biologi-
cal evaluation of nucleoside analogs capable of delivering the
by our discovery that vidarabine was 3- to 5-fold more active
against vaccinia and cowpox viruses than cidofovir in plaque
corresponding nucleotides inside cells.1 1-Beta-
D-arabinofurano-
reduction assays.10 Cidofovir is
a broad spectrum antiviral
syladenine (vidarabine or ara-A) is an antiviral drug with activity
against herpes viruses, poxviruses, and certain rhabdoviruses, hep-
adnarviruses, and RNA tumor viruses.2–4 Vidarabine also is active
against vaccinia virus both in vitro5 and in vivo.6 However, it is
more toxic and less metabolically stable than other current antiv-
irals such as acyclovir and ganciclovir; further it is poorly soluble
with low oral bioavailability. It is readily deaminated by adenosine
deaminase (ADA) to ara-hypoxanthine (ara-H),7 which possesses
some antiviral activity but is at least 10-fold less potent than vidar-
abine.6–8 Adenosine deaminase (ADA) is a cytosolic enzyme that
participates in purine metabolism where it degrades either adeno-
sine or 20-deoxyadenosine to inosine or 20-deoxyinosine, respec-
tively. Further metabolism of these deaminated nucleosides leads
to hypoxanthine. ADA also degrades vidarabine to ara-H by same
mechanism.7
agent,11–13 that is, limited in its usage because of nephrotoxicity
and poor oral bioavailability (ꢀ2% in humans)14–16 and for which
prodrugs have been developed.17 Furthermore, we found that the
activity of vidarabine against these viruses was enhanced approx-
imately 10-fold when combined with 20-deoxycoformycin (pento-
statin, a potent inhibitor of ADA), thus providing significant
superiority to cidofovir. Based on these results and earlier studies
on 50-substituted vidarabine analogs, we hypothesized that mini-
mizing the conversion of vidarabine to its hypoxanthine analog
could yield a significantly more potent anti-pox virus agent. With
this goal in mind, we have developed a prodrug strategy that pro-
tects the vidarabine from metabolic conversion by making 50-ami-
no acid esters and 50-phosphoramidates of the drug. Further, our
rationale includes the design of prodrugs that increase aqueous
solubility over that of the parent drug and also increase the poten-
tial for membrane transport by the dipeptide intestinal
transporter.
Our current interest in prodrugs of vidarabine was triggered by
the report of the activity of vidarabine against cowpox virus9 and
In order to realize the efficient high throughput synthesis of 50-
amino acid ester and 50-(phenyl methoxyamino acid)-phosphate
derivatives of vidarabine in large quantities (100 mg for each), it
* Corresponding author. Tel.: +1 734 663 4233.
E-mail address: wshen@tsrlinc.com (W. Shen).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.031

W. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 792–796
793
is crucial to selectively protect the 20 and 30 hydroxyl groups of the
arabinoside residue. Such blocking groups have to be easily and
quickly removed under non-basic conditions to prevent concomi-
tant cleaving of the acyl groups in the phosphate or amino acid es-
ter moiety. After evaluation of a range of protecting groups,
including benzoate and acetate, the final candidate for protection
of the 20, 30 hydroxyl positions was the levulinate group. The levu-
linate group can survive the synthesis conditions for these pro-
drugs and can be easily removed by treating with 1 ml of 2 M
hydrazine hydrate in pyridine-acetic acid buffer for 10 min,18,19
conditions under which normal esters are not cleaved.20,21 In addi-
tion, the levulinate is less prone to migration between adjacent hy-
droxyl groups on the sugar residue than other ester protection
group such as benzoate and acetate.22,23 Finally, the use of flash
chromatography during the purification process was minimized
during the parallel synthesis of a representative prodrug library,
which significantly improved the overall efficiency of the
synthesis.
NH2
N
NH2
N
N
N
N
N
Si
HO
O
HO
N
N
O
The typical procedure for synthesis of 20 and 30 protected vidar-
abine is depicted in Scheme 1. First, selective protection of the
50-OH of vidarabine was readily accomplished with tert-butyldi-
methylsilyl chloride in the presence of imidazole in DMF. The
resulting 50-O-TBDMS-vidarabine 2 was purified with liquid–liquid
extraction between water and ethyl acetate, giving a 90% yield.
Compound 2 was then acylated with levulinic anhydride, which
was generated in situ from levulinic acid with DCC in the presence
of DMAP as catalyst to produce the fully blocked 50-O-TBDMS-20,30-
dilev-ara A 3. Importantly, the exocyclic amine of the adenine moi-
ety was not levulinated as long as the reaction time was less than
two hours. This regioselectivity allowed for the avoidance of pro-
tection and deprotection steps of the exoclyclic amine group. Li-
quid–liquid extraction between saturated ammonium chloride
and ethyl acetate was performed, followed by silica gel flash chro-
matography to purify the product. Selective removal of the 50-
TBDMS group was readily achieved with a mixture of TBAF/acetic
acid (1:2 mole ratio) in tetrahydrofuran. In accordance with the
observations of other workers,24 we noticed that there were some
HO
a
O
2
1
HO
HO
Vidarabine
b
O
N
O
NH2
NH2
N
N
N
N
O
O
O
Si
O
N
N
4
O
N
HO
c
O
O
O
O
O
O
3
O
O
Scheme 1. Reagents and condition: (a) TBSCl and Im./DMAP DMF; (b) levulinic
acid, DCC, DMAP in ethyl acetate; and (c) TBAF/acetic acid (1:2 mole ratio) in
tetrahydrofuran.
O
O
NH2
N
NH2
N
BocHN
R
N
N
N
N
O
O
O
N
HO
O
O
O
O
O
N
R:
O
a
O
O
7 b, j leucine
7 a,i
valine
phenylalanine
serine
7 d,
7 f,
l
isoleucine
7 c,k
5
O
n
O
7 e, m lysine
7 g,o
O
b
aspartic acid
NH2
N
NHBoc
O
NH2
N
NH2
N
N
N
R
N
N
N
N
HO
O
N
N
O
O
N
HO
O
O
O
HO
O
O
H
H
HO
6
O
OH
OH
H2N
c
OH
7 h
H2N
HO
O
7 p
NH2
O
H
NH2
NH2
N
NH2
N
N
N
N
H
R
HO
O
N
O
HO
O
N
N
R
O
O
HO
L-amino acid ester
7 i -p
HO
D- amino acid ester
7 a -h
Scheme 2. Reagents and condition: (a) alpha N-Boc D- and L-amino acid, DCC, DMAP DMF; (b) 1 ml of 2 M hydrazine hydrate in pyridine–acetic acid buffer; and (c) removal of
protection group from amino acid moieties.

794
W. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 792–796
O
O
P
O
TEA
Cl
-Cl+H3N
Cl
O
P
O
+
OMe
O
DCM
Cl
HN
R
8
OMe
R
NH2
N
O
O
NH2
N
N
N
O
N
N
O
O
O
O
O
N
P
HO
O
N
HN
Cl
O
O
O
P
O
O
N-methyl imidazole
+
OMe
R
O
HN
THF
O
O
MeO
O
R
O
O
O
9
4
8
a
NH2
NH2
R:
O
N
N
O
O
Me Me
N
O
N
P
N
P
HN
N
HN
O
N
N
O
HO
O
HO
R
H
O
R
H
O
OMe
10 a
OMe
O
HO
HO
10 b
Scheme 3. Reagents and condition: (a) one milliliter of 2 M hydrazine hydrate in pyridine–acetic acid buffer.
acyl migration (30–50) of the levulinyl group in these arabinoside
derivatives when acetic acid was absent during the treatment with
TBAF. Addition of acetic acid in the reaction system can prevent the
migration from occurring. The 20,30-dilevulinyl vidarabine (4) ob-
tained was purified with silica gel flash chromatography eluting
with 8% methanol in DCM. The total yield from vidarabine to
20,30-dilev vidarabine was 74%.
Table 1
Antiviral activity of amino acid prodrugs of vidarabinea
b
b
Promoiety (Compound)
EC50
Vaccinia
(l
M)
CC50
HFF
(lM)
Cowpox
Vidarabine (1)c
6.2
1.0
2.9
4.5
3.0
3.3
4.1
4.8
2.3
1.8
2.5
3.5
12.6
1.4
3.5
2.5
2.5
3.0
8.0
4.0
4.0
3.5
4.0
25
>100
90
Vidarabine + dCFc
D
-ile (7c)
-ile (7k)
-leu (7b)
-leu (7j)
-val (7a)
-val (7i)
-phe (7d)
-phe (7l)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
After obtaining sufficient quantity of 20,30-dilevulinyl vidara-
bine, a parallel synthesis strategy was applied to synthesize repre-
L
D
sentative libraries of 50-
D
- or
and 50-[phenyl (amino acid methyl ester)]-phosphate derivatives
(Scheme 3) of vidarabine. The protected - or -amino acids were
L-amino acid derivatives (Scheme 2)
L
D
L
D
L
D
coupled with protected vidarabine using DCC as a coupling reagent
and DMAP as a catalyst (Scheme 2). The fully blocked products 5
were isolated with liquid–liquid extraction between saturated
ammonium chloride and ethyl acetate and dried with Na2SO4. After
removal of solvent, the crude products were treated with 1 ml of
2 M hydrazine hydrate in pyridine–acetic acid buffer for 10 min
to produce the uncharged species 6. The excess hydrazine was
eliminated through reaction with pentane-2,4-dione for another
10 min. After evaporating the volatile components, the residue
was dissolved in EtOAc, washed with saturated ammonium chlo-
ride, water and brine. The organic layer was dried over Na2SO4, fil-
tered and evaporated to dryness. Removal of the protection groups
on the amino acid promoieties generated the final products 7a–p,
which were purified by preparative HPLC.
L
D-a-asp (7g)
D-b-asp (7h)
a
Fifty percent inhibitory concentration by plaque assay against the two viruses
grown in human foreskin fibroblasts (HFF’s).
b
Visual cytotoxicity determined in HFF’s not affected by virus replication
expressed as 50% inhibitory concentrations.
c
Data for vidarabine and vidarabine plus
1 lM deoxycoformycin (dCF) are
averages from four to nine experiments. Prodrug data for vaccinia virus are aver-
ages of two to four experiments except for the asp prodrugs where data are from a
single experiment. Likewise data for cowpox are from one experiment.
Phosphoramidate prodrugs were synthesized according to the
method described in Scheme 3. The phenyl (amino acid methyl es-
ter) phosphochloridates 8, were obtained based on modified liter-
Table 2
Direct uptake of L-val and L-Ile vidarabine prodrugs in HeLa/hPEPT1 and HeLa cells
ature methodology.25 Compound
8 was then coupled with
Promoiety
(Compound)
HeLa/hPEPT1 (nmol/
mg/45 min)
HeLa control (nmol/
mg/45 min)
hPEPT1
control
protected vidarabine 4 in the presence of N-methyl imidazole
and THF to yield the blocked phosphoramidate vidarabine pro-
drugs such as 9. The resultant product 9 was purified by silica
gel flash chromatography with 6% methanol in DCM as eluent.
After treatment with 1 ml of 2 M hydrazine hydrate in pyridine-
acetic acid buffer for 10 min, the same workup method for com-
pound 7 was applied and preparative HPLC was used to obtain
L
-val (7i)
-ile (7k)
80.3 1.1a
202 4.8
221 8.1
3.9 0.3
7.5 0.2
11.6 0.5
20.7
26.9
19.0
L
Valacyclovirb
a
All data are presented as means and standard deviations from three
experiments.
b
Valacyclovir was used as a positive control for hPEPT1 carrier mediated uptake.

W. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 792–796
795
the pure products 10a and b. We used both
D
- and
L
-amino acids to
ported previously for HSV-1,26 addition of the ADA inhibitor deox-
ycoformycin increased the activity of vidarabine against the
poxviruses approximately 5- to 10-fold. Also as shown in Table 1,
the amino acid prodrugs were more effective against these viruses
than was the parent drug. In some experiments some prodrugs
were almost as active as vidarabine plus deoxycoformycin. The
apparently greater activity of the prodrugs compared to vidarabine
may mean that the prodrugs are not substrates for adenosine
deaminase and thereby protect the parent drug from deamination
before hydrolysis to vidarabine in cell culture. Alternatively or in
addition, this higher potency could be a result of more rapid pen-
etration of the prodrugs into infected cells or the prodrugs could
be inhibitors of ADA.
build the representative libraries for compounds 7 and 10. The de-
tailed synthesis procedures and analytical data can be found in the
Supporting Material.
Vidarabine exhibited good activity in cell culture against two
small pox related viruses, vaccinia and cowpox (Table 1). As we re-
Table 3
Stability of vidarabine and its prodrugs in biological homogenates and plasma from
rat
Promoiety (Compound)
T1/2 (hours)
Liver homogenate
Intestinal homogenate
Plasma
Absorption of vidarabine from the intestine is very poor. In or-
der to achieve sufficiently high plasma levels the drug should be gi-
ven intravenously or through ocular delivery. The recommended
intravenous injection dose is 10–15 mg/kg daily. Due to the poor
water solubility, vidarabine must be administered in a large vol-
ume of an appropriate intravenous infusion fluid (e.g., glucose
5%). The daily dose is slowly infused in a 12–24 h period for at least
10 days.27 The vidarabine prodrugs are being developed to over-
come the biopharmaceutical limitations of vidarabine, including
poor solubility, poor intestinal transport and rapid metabolism of
the parent vidarabine to ara-H. Vidarabine has a reported solubility
of ꢀ0.47 mg/ml in water, with increasing solubility at lower pH.28
The amino acid prodrugs described in this report are all freely sol-
Vidarabine (1)
0.05
7.90
0.58
0.04
0.03
0.59
0.27
0.30
Sta
0.25
0.31
0.06
0.06
0.05
0.10
<0.017
0.03
0.46
5.91
Sta
0.43
1.61
3.58
nab
D
-ile (7c)
-ile (7k)
-leu (7b)
-leu (7j)
-phe (7d)
L
D
L
nab
D
nab
L
L
-phe (7l)
-val (7j)
nab
0.13
10.26
0.35
0.30
D
-val (7a)
L
P-
P-
valc (10a)
valc (10b)
18.32
D
Sta
a
b
c
St means stable.
na means not analyzed.
Phosphoramidate prodrug.
Deamination of vidarabine
400
350
300
250
200
150
100
50
Vidarabine
Ara-H
0
0
20
40
60
80
100
Time, min
Percentage remaining of Vidarabine and prodrug after
incubation with adenosine deaminase
95
100
90
80
70
60
50
40
30
20
10
0
20.8
Vidarabine
5'-D-valyl ara A
Figure 1. Deamination of vidarabine and its prodrugs by adenosine deaminase1. Top: Concentration (
lM) profiles of the disappearance of vidarabine and the appearance of
Ara-H in the presence of adenosine deaminase. Bottom: Percent vidarabine or prodrug remaining following incubation with adenosine deaminase1 for 90 min.

796
W. Shen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 792–796
Table 4
Acknowledgement
Summary of permeability data for vidarabine and selected prodrugs
Compounds
Caco-2 permeability cm/s ꢁ 106
0.083
0.601
1.026
0.668
Fold increase
This work was supported by a SBIR Grant 1R43AI071400 from
the National Institutes of Health.
Vidarabine (1)
—
7.2
12.4
8.05
D-val (7a)
L
-ile (7k)
-phe (7l)
Supplementary data
L
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.12.031.
uble in water and have greatly improved solubility at physiological
pH, with solubilities >10 mg/ml. Based upon our previous findings
with amino acid nucleoside prodrugs,29 it was anticipated that the
amino acid prodrugs of vidarabine would be substrates for the
dipeptide intestinal transporter, PEPT1. To test this, the uptake of
References and notes
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b) were the most stable followed by the
then the -amino acid prodrugs. Hydrolysis to parent was more ra-
pid in the liver homogenates for all prodrugs, except the -valyly
D-amino acid prodrugs
L
D
phosphoramidate vidarabine, which was stable in both homoge-
nates. However, in general, the phosphoramidate prodrugs were
less stable than their amino acid counterparts in plasma. Stability
to ADA, which is the major metabolic enzyme for vidarabine and
which converts it to ara-H was also examined. While vidarabine
was rapidly hydrolyzed with a commensurate rise in the concen-
tration of the deaminated product, ara-H (Fig. 1 top), it was found
that substitution at the 50-OH group made the drug resistant to
deamination by ADA (Fig. 1 bottom),36,37 consistent with past re-
ports on 50-OH prodrugs of vidarabine.8,26
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The transepithelial transport of some of the prodrugs through
Caco-2 monolayers was evaluated as previously described.38 In
these experiments, transport studies were performed 21 days
post-seeding. The assay was initiated by adding drug transport
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29. Vig, B. S.; Lorenzi, P. J.; Mittal, S.; Landowski, C. P.; Shin, H.-C.; Mosberg, H. I.;
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cose, 5 mM MES, 1 mM CaCl2, 1 mM MgCl2, 150 mM NaCl, 3 mM
KCl, 1 mM NaH2PO4) to the apical chamber of the Caco-2 insert.
Two hundred microliter aliquots were withdrawn from the basolat-
eral chamber at predetermined intervals and replaced with fresh
HEPES, pH 7.4, buffer. The epithelial integrity of representative cell
monolayers was assessed by monitoring transepithelial resistance.
As seen in Table 4, the permeabilities of all tested prodrugs were en-
hanced by at least 7-fold comparing with the permeability of vidar-
abine. Considering that the prodrugs would be substrates for the
dipeptide intestinal transporter, PEPT1 (Table 2) and those trans-
porters are much more highly expressed in the small intestinal
membranes than in Caco-2 membranes, we anticipate significant
increases of vidarabine and/or its prodrug in in vivo studies.
These data support our hypothesis that substitution at the 50-
OH group can result in both enhanced uptake of vidarabine or its
prodrugs and protection from metabolism by ADA. The next phase
of the work will be to examine the in vivo oral bioavailability of the
prodrugs and parent compounds and ultimately their effectiveness
in animal models of pox viruses.
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