The application of phosphoramidate protide technology to acyclovir confers anti-HIV inhibition

Article abstract of DOI:10.1021/jm9007856

Recently, it has been reported that phosphorylated acyclovir (ACV) inhibits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a cell-free system. To deliver phosphorylated ACV inside cells, we designed ACV monophosphorylated derivatives using ProTide technology. We found that the L-alanine derived ProTides show anti-HIV activity at noncytotoxic concentrations; ester and aryl variation was tolerated. ACV ProTides with other amino acids, other than L-phenylalanine, showed no detectable activity against HIV in cell culture. The inhibitory activity of the prodrugs against herpes simplex virus (HSV) types -1 and -2 and thymidine kinase-deficient HSV-1 revealed different structure-activity relationships but was again consistent with successful nucleoside kinase bypass. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. ProTides showing diminished carboxypeptidase lability translated to poor anti-HIV agents and vice versa, so the assay became predictive.

Full text of DOI:10.1021/jm9007856

5520 J. Med. Chem. 2009, 52, 5520–5530
DOI: 10.1021/jm9007856
The Application of Phosphoramidate Protide Technology to Acyclovir Confers Anti-HIV Inhibition
Marco Derudas,† Davide Carta,† Andrea Brancale,† Christophe Vanpouille,‡ Andrea Lisco,‡ Leonid Margolis,‡ Jan Balzarini,§
and Christopher McGuigan*,†
†Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3NB, U.K., ‡Program in Physical Biology, Eunice Kennedy Shriver National
Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, and §Rega Institute for Medical
Research, Katholieke Universiteit, Leuven B-3000, Belgium
Received June 2, 2009
Recently, it has been reported that phosphorylated acyclovir (ACV) inhibits human immunodeficiency
virus type 1 (HIV-1) reverse transcriptase in a cell-free system. To deliver phosphorylated ACV inside
cells, we designed ACV monophosphorylated derivatives using ProTide technology. We found that the
L-alanine derived ProTides show anti-HIV activity at noncytotoxic concentrations; ester and aryl
variation was tolerated. ACV ProTides with other amino acids, other than L-phenylalanine, showed no
detectable activity against HIV in cell culture. The inhibitory activity of the prodrugs against herpes
simplex virus (HSV) types -1 and -2 and thymidine kinase-deficient HSV-1 revealed different structure-
activity relationships but was again consistent with successful nucleoside kinase bypass. Enzymatic and
molecular modeling studies have been performed in order to better understand the antiviral behavior of
these compounds. ProTides showing diminished carboxypeptidase lability translated to poor anti-HIV
agents and vice versa, so the assay became predictive.
Introduction
ACV inhibits HIV RT acting as a chain terminator.3 Follow-
ing these results, it is evident that the anti-HIV activity can
only occur upon ACV monophosphate (ACV-MP) formation
which requires HHV coinfection. ACV-MP itself can not be
used as efficient anti-HIV chemotherapeutic agent to bypass
the first limitingphosphorylation step becauseofits instability
inbiologicalmedia andits poor efficiencyof diffusion through
intact cell membranes. A suitable strategy to overcome these
limitations would consist of masking the negative charges of
the monophosphate with lipophilic groups. In this regard, the
phosphoramidate ProTide technology has been developed
and successfully applied to a range of nucleosides of antiviral
and anticancer interest.6-9 The structural motif of this app-
roach consists of masking the nucleoside monophosphate
with an aryl moiety and an amino acid ester. Cell entry then
apparently occurs by passive diffusion. Once inside the cell,
the phosphoramidate prodrug is activated and converted to
the monophosphorylated ACV (Figure 1).10 The first step
involves an enzymatic hydrolysis of the amino acid ester
moiety mediated by an esterase- or carboxypeptidase-type
enzyme followed by spontaneous cyclization and subsequent
spontaneous displacement of the aryl group and opening of
the unstable ring mediated by water. The last step before
release of the ACV monophosphate involves a hydrolysis of
the P-N bond mediated by a phosphoramidase-type enzyme.
The phosphoramidate ProTide approach has been already
successfully applied to ACV, demonstrating its ability to
bypass the thymidine kinase deficiency of HSV-1 and -2 and
varicella zoster virus strains resistant to ACV.11
Human immunodeficiency virus (HIVa) belongs to the
retroviradae family and causes the acquired immunodefi-
ciency syndrome (AIDS). A variety of different compounds
have been developed for the treatment of HIV, and currently
25 drugs have been approved for clinical use including nucleo-
side reverse transcriptase inhibitors (NRTIs), non-nucleoside
reverse transcriptase inhibitors (NNRTIs), protease inhibi-
tors (PI), a viral fusion inhibitor (FI), a CCR-5 coreceptor
inhibitor, and a viral integrase (IN) inhibitor.1
Because of the rapid development of drug resistance as well
as to the toxicity shown by these drugs,2 novel anti-HIV
agents are needed. Diverse structures are sought to address
the constant threat of viral resistance.
In this context, recently it has been reported how the
antiherpetic drug acyclovir (ACV, 1, Figure 2) inhibits HIV
upon human herpesvirus (HHV) coinfection in tissue cul-
tures.3 This activity was found to be correlated with the
phosphorylation of the parent drug to the monophosphate
form mediated by HHV-encoded kinase(s). HIV does not
encode an enzyme that recognizes ACV as a substrate for this
activation (phosphorylation) step, hence the need for the
HHV coinfection for activity. The subsequent phosphoryla-
tions to the di- and triphosphate derivatives may be mediated
by cellular guanosine monophosphate kinase and nucleoside
diphosphate kinase, respectively.4,5 In its triphosphate form,
*To whom correspondence should be addressed. Phone: þ44 29
20874537. Fax: þ44 29 20874537. E-mail: mcguigan@cardiff.ac.uk.
a Abbreviations: ACV, acyclovir; HIV, human immunodeficiency
virus; HSV, herpes simplex virus; NNRTIs, non-nucleoside reverse
transcriptase inhibitors; PI, protease inhibitor; FI, fusion inhibitor;
HHV, human herpes virus; ACVMP, acyclovir 50-monophosphate;
DMF, N,N-dimethylformamide.
In this paper, we present the synthesis and initial biolo-
gical evaluation of a novel series of ACV ProTides
(Figure 2). The ProTide moiety has three different change-
able parts: the aryl moiety, the amino acid, and the ester. In
the first part of this study, we have chosen L-alanine as the
r
pubs.acs.org/jmc
Published on Web 07/31/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5521
Figure 1. Proposed activation pathway of the acyclovir ProTides.
Scheme 1a
a Reagents and conditions: (i) POCl3, anhydrous TEA, anhydrous
Et2O, -78 °C, 1 h then rt, overnight.
Figure 2. ACV and its ProTides.
Scheme 2a
amino acid, as it has shown previously an optimal biologi-
cal profile,12 varying the other two components. For the
aryl moiety we considered phenol, naphthol, and p-fluoro-
phenol and as the ester moiety methyl, ethyl, n-propyl, iso-
propyl, tert-butyl, and benzyl and combinations thereof.
All of these combinations allowed us to extensively vary
the LogP for these compounds and to study how this
variation can influence the antiviral activity. Moreover, it
has been previously reported how the substitutions can
influence the bioactivation of the ProTide; for example,
naphthol has shown an enhancement of activity against a
panel of cancer cell lines for phosphoramidates6 and the
tert-butyl ester showed a lack of biological activity due to
the poor bioactivation of the bulky moiety. Following the
results for these derivatives, different amino acids have been
considered including L-valine, L-leucine, L-isoleucine, L-
proline, glycine, and the non-natural D-alanine, D-valine,
and dimethylglycine. Moreover, some intermediate-prote-
cted (N2-DMF)-ACV ProTides have been biologically
evaluated.
a Reagents and conditions: (i) anhydrous TEA, anhydrous DCM,
-78 °C, 30 min to 1 h, then rt, 30 min to 4 h.
To improve the solubility of ACV in THF, used as ideal
solvent for the coupling reaction, the 2-amino was protected
using dimethylformamide dimethyl acetal (Scheme 3). How-
ever, compound 66 is not completely soluble in THF but the
solubility was improved sufficiently to carry out the reaction.
The final coupling of the nucleoside was performed using an
excess of the appropriate phosphorochloridate (1.50-4.00
equiv) in the presence of tBuMgCl (2 equiv). Because of the
reactivity problem, the use of N-methylimidazole (NMI),
following the Van Boom procedure,14 was used for the
synthesis of the L-proline (22) and glycine (23) derivatives.
Moreover, a mixture of THF/pyridine (3/2) was used as a
solvent to improve the solubility of N2-DMF-ACV.
Chemistry
The compounds have been synthesized following the pro-
cedure reported by Uchiyama13 using tert-butylmagnesium
chloride (tBuMgCl) as a coupling reagent and using THF as a
solvent in most of the cases.
Aryl phosphorodichlorophosphates 26 and 27 have been
synthesized, coupling respectively 1-naphthol (24) or p-fluor-
ophenol (25) with POCl3 in the presence of Et3N (1equiv)
(Scheme 1), while phenyl dichlorophosphate (28) was com-
mercially available. The coupling with the appropriate amino
acid ester salt (29-44) has been performed in the presence of
Et3N (2 equiv) (Scheme 2), giving the final product (45-65) as
an oil which was, in most of the cases, purified by column
chromatography.
The deprotection of the dimethylformyl DMF derivative
was initially carried out by refluxing the compound in
1-propanol (Scheme 3). However, because of a transesterifica-
tion during the synthesis of 2, obtaining compound 3, the
solvent was changed to 2-propanol, obtaining the desired
compounds (2, 4-23).
All the compounds were obtained as a mixture of
two diastereoisomers confirmed by the presence of two peaks
in the 31P NMR, with the exception of the glycine and

5522 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Derudas et al.
Scheme 3a
t
a Reagents and conditions: (i) dimethylformamide dimethyl acetal, anhydrous DMF, rt, 1 day; (ii) BuMgCl, THF, rt, overnight or NMI, THF/
pyridine=3/2, rt, overnight; (iii) 1-propanol, reflux, for 18 h or 2-propanol, reflux, 24-96 h.
Table 1. Anti-HSV Activity for ACV and Its Protides
antiviral activity EC50a (μM)
cytotoxic/cytostatic activity (μM)
compds
aryl
amino acid
ester
CLogP
HSV-1
HSV-2
HSV-1 TK-
MCCb (Hel)
IC50c (Hel)
2
Naph
Naph
Naph
Naph
Naph
Naph
Ph
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
D-Ala
DMG
DMG
Phe
Bn
nPr
Me
Et
2.06
1.41
0.35
0.88
1.59
1.19
-0.82
0.89
0.02
1.11
0.89
2.37
1.20
2.31
1.82
1.28
1.81
1.28
2.35
2.35
2.82
0.58
-2.42
2 ( 0
1.4 ( 0.8
1.9 ( 1.6
10 ( 2.1
9.5 ( 0.7
50
10 ( 2.1
16 ( 5.7
79 ( 29
32 ( 18
>100
g20
g50
20
68
3
5.5 ( 2.1
16 ( 5.7
32 ( 25
>100
4
>50
>100
>100
>100
>100
ND
91
5
>150
>50
>50
6
tBu
iPr
Me
Bn
iPr
Bn
Bn
Bn
Bn
Bn
Bn
Me
Et
7
15 ( 7.1
20 ( 0
8 ( 5.7
10 ( 0
0.9 ( 0.1
2 ( 0
2.4 ( 0
1.4 ( 0.85
17 ( 4.2
2 ( 0
10 ( 0
16 ( 5.7
4 ( 0
g45
8
79 ( 29
15 ( 7.1
27 ( 25
1.5 ( 0.7
23 ( 16
3.2 ( 1.1
5.5 ( 2.1
g100
>100
>50
>50
9
Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ACV (1)
8.5 ( 0.7
0.5 ( 0
1.4 ( 0.8
1.6 ( 1.1
0.8 ( 0.0
8 ( 5.7
0.85 ( 0.2
>100
>100
ND
ND
>100
ND
87
p-F-Ph
Ph
>100
>100
>50
Naph
Ph
Ph
>100
>50
Ph
L-Val
L-Val
L-Val
D-Val
L-Leu
L-Ile
7.5 ( 6.4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ND
ND
ND
ND
ND
ND
>100
ND
ND
Naph
Naph
Naph
Ph
>100
51 ( 9.2
>100
32 ( 18
>100
42 ( 3.5
>100
Me
Bn
Bn
Bn
Bn
0.8 ( 0.07
1.1 ( 0.4
>100
0.7 ( 0
1.1 ( 0.4
>100
1.4 ( 0.8
1.4 ( 0.8
>100
Ph
Ph
L-Pro
Gly
Ph
3
0.4
0.8
0.2
9
50
a 50% Effective concentration, or compound concentration required to inhibit virus-induced cytopathicity by 50%. b Minimal cytotoxic concentra-
tion, or compound concentration required to cause a microscopically visible alteration of cell morphology. c 50% Inhibitory concentration, or
compound concentration required to inhibit cell proliferation by 50%. ND=not determined
dimethylglycine derivatives, due to the absence of a chiral
center, and L-proline, for which we were able to isolate only
one diastereoisomer.
derivatives, showed anti-HSV activity in the submicromolar
range, while the majority of the compounds showed an
activity in the range of ca. 1-30 μM. Compound 6 having
the bulky t-butyl group as ester moiety did not show any
marked activity (g50 μM) against the HSV strains. ACV has
been evaluated against thymidine kinase-deficient HSV-1
showing a dramatic loss of activity (>100-fold) (EC50:
50 μM). Interestingly, several of the ProTides showed sig-
nificant retention of activity, demonstrating a successful
bypass of the first phosphorylation step (i.e., compounds
11, 13, 20, 21). Notably, none of the ACV ProTides showed
appreciable cytostatic/cytotoxic activity despite the potential
loss of antiviral selectivity that could follow from viral
nucleoside kinase bypass.
Biological Results
Anti-HSV Activity. The activity of the compounds were
evaluated against three different strains of HSV including
HSV-1 (KOS), HSV-2 (G), and thymidine kinase-deficient
HSV-1 (ACVR).
Native ACV showed submicromolar (EC50: 0.4 μM and
0.2 μM) activity against respectively HSV-1 and HSV-2
(Table 1) but was inactive against TK-deficient HSV-1
(EC50: 50 μM). The ACV ProTides did not show increased
activity against HSV-1 and HSV-2 compared to the parent
compound. Only two compounds (11 and 20), respectively
the p-fluorophenyl-L-Ala-OBn and the phenyl-L-Leu-OBn
Anti-HIV Aactivity. The ACV ProTides have also been
evaluated against HIV-1 and HIV-2 in CEM and against

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5523
Table 2. Anti-HIV Activity of the ACV ProTides and ACV
antiviral activity EC50a (μM)
cytostatic activity (μM)
compds
aryl
amino acid
ester
HIV-1 CEM
HIV-2 CEM
HIV-1 MT-4
IC50bCEM
CC50c (MT-4)
IC50b (MT-4)
2
Naph
Naph
Naph
Naph
Naph
Naph
Ph
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
L-Ala
D-Ala
DMG
DMG
Phe
Bn
nPr
Me
Et
15 ( 14
6.6 ( 5.6
10 ( 7.9
12 ( 9.8
>100
6.2 ( 5.4
17 ( 4.6
16 ( 14
>100
>20
8.9 ( 6.3
24 ( 30
13 ( 6.4
42 ( 13
>100
12 ( 0.71
26 ( 8.5
11 ( 4.9
>100
>20
0.8
10
17
22
ND
ND
>150
ND
3
4
4.7 ( 2.1
1.7 ( 0.8
>150
5.4
57
>150
>150
>150
>150
ND
18.7 ( 3.2
12 ( 5.3
>150
72.5
5
32 ( 7.8
>100
36 ( 15
67 ( 7.8
42 ( 11
>100
76 ( 13
g250
>100
>100
42
6
tBu
iPr
Me
Bn
iPr
Bn
Bn
Bn
Bn
Bn
Bn
Me
Et
7
8
15
ND
9
Ph
Ph
5.7 ( 1.6
>150
ND
ND
ND
7
>150
>150
ND
33.8 ( 10.6
>150
ND
10
11
12
13
14
15
16
17
18
19
20
21
22
23
77d
78d
81d
86d
ACV (1)
p-F-Ph
Ph
>250
g100
>250
79 ( 30
>100
34 ( 24
>50
ND
ND
ND
ND
Naph
Ph
Ph
>100
26 ( 11
>50
>150
ND
ND
>150
ND
ND
16
Ph
L-Val
L-Val
L-Val
D-Val
L-Leu
L-Ile
ND
ND
ND
ND
0.8
g100
>100
>100
>100
>20
Naph
Naph
Naph
Ph
>100
>100
>100
>20
>100
>100
>100
>20
ND
ND
ND
ND
Me
Bn
Bn
Bn
Bn
Bn
Bn
Me
Bn
ND
17
ND
>150
ND
ND
Ph
Ph
>20
>20
>20
>20
ND
ND
ND
15
ND
ND
ND
L-Pro
Gly
>100
>100
40 ( 2.8
>100
>100
45 ( 0.0
>250
Ph
Naph
Ph
>100
>20
>100
>20
ND
45
ND
140
DMG
DMG
L-Val
L-Pro
>100
>100
>20
>100
>100
>20
70
>150
>150
90
>150
>150
>150
>250
Naph
Ph
>150
30
>250
>250
>250
>250
a 50% Effective concentration, or compound concentration required to inhibit virus-induced cytopathicity by 50%. b 50% Cytotoxic concentration,
or compound concentration required to decrease the viability of the cell cultures by 50%. c 50% Inhibitory concentration, or compound concentration
required to inhibit cell proliferation by 50%. ND = not determined. d N2-DMF-ACV.
HIV-1 in MT-4 cell cultures and in HIV-infected tonsillar
tissues ex vivo (Table 2).
(23), did not show appreciable activity in CEM. These results
are in agreement with previous reports for other nucleosides,
While parent ACV was inactive, in most of the L-alanine
derivatives (2-11), with the exception of 6, 10, and 11,
showed activity (EC50) in a range of 6.2-17 μM against
HIV-1 (CEM) and in a range of 8.9-42 μM against HIV-2
(CEM) (Table 2). Similar results were obtained when these
compounds were applied to the HIV-1-infected MT-4 cell
cultures. In these cells, parental ACV was inactive whereas
HIV was suppressed by all the tested compounds with EC50’s
in the range of 0.8-30 μM, with the exception of 6, 10, 78,
and 81 (Table 2). The antiviral activity of the ACV ProTides
was confirmed in HIV-infected tonsillar tissue ex vivo. The
compounds 4, 5, and 9 suppressed HIV replication with
EC50’s in a range of 0.1-0.6 μM (data not shown). These
findings indicate that the ACV ProTide approach can also be
successfully applied to HIV-infected tissue.
No clear-cut structure-activity relationship could be ob-
served with regard to the nature of the aryl moiety nor the
alaninyl ester moiety in terms of eventual antiviral activity of
the ProTide derivatives. The lack of activity obtained for the
t-butyl analogue (6) is in agreement with the previous report
and with the enzymatic experiment to be discussed later. All
of these compounds showed an antiproliferative effect (on
CEM or MT-4 cell cultures) in a range between 17 and
76 μM.
in which the substitution of the
natural L-amino acids gave loss (∼L1-0a-latnoin1e00w-fiotlhd)doifffearnetnit-
viral activity.15 However, in the case of dimethylglycine, this
result is quite surprising as this variation led usually to a
retention of anti-HIV activity compared to the L-alanine
derivatives.16
Interestingly, in MT-4 cell cultures, the amino acid mod-
ification seems to be tolerated, in fact compounds 14 (DMG)
and 20 (L-Leu), showed respectively an anti-HIV-1 activity of
7 and 0.8 μM.
Moreover, in MT-4, the N2-DMF-protected ACV Pro-
Tides (77, 78, and 86) showed activity against HIV-1 (EC50’s:
15, 70, and 30, respectively), indicating that this kind of
substitution may be tolerated. However, in the case of CEM,
these compounds did not show any inhibitory activity.
From these results, it is possible to conclude that the
amino acid L-alanine is optimal for the anti-HIV activity of
the ACV ProTides. Neither D-alanine nor glycine can effi-
ciently substitute for L-alanine nor can bulkier amino acids.
In contrast to the structural requirement for anti-HIV
activities, the anti-HSV activity tolerates liberal amino acid
variation. This may reflect different substrate specificities
and/or different intracellular levels of the necessary activat-
ing enzymes. It should indeed be noticed that the HIV
assays are performed in rapid proliferating lymphocyte cell
cultures (generation time ∼ 24 h), whereas the antiherpetic
assays are carried out in confluent fibroblast monolayer
(nonproliferating) cell cultures. Thus, the different cell-type
and cell-cycle conditions between both assay models can
result in different prodrug activation modalities that may
With regard to the amino acid modifications, we found
that, besides the L-alanine ProTides, only the phenylalanine
derivative (15) had activity against HIV-1 (26 μM) and HIV-
2 (34 μM). All other derivatives, including D-alanine (12),
dimethylglycine (13 and 14), L-valine (16-18), D-valine (19),
L-leucine (20), L-isoleucine (21), L-proline (22), and glycine

5524 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Derudas et al.
Figure 3. Carboxypeptidase-mediated cleavage of compound 9, monitored by 31P NMR.
Figure 4. Carboxypeptidase-mediated cleavage of compound 10, monitored by 31P NMR.
explain the differential antiviral activities of the ACV Pro-
Tides.
Enzymatic Studies. The mechanism of activation of the
ProTides involves a first enzymatic activation step mediated
by carboxypeptidase-type enzyme(s), which hydrolyze the
ester of the amino acid moiety (Figure 1).
enzymatic study using carboxypeptidase Y following the
conversion by 31P NMR. Of three different L-alanine deri-
vatives (9, 6, and 10), the first one is active vs HIV and the
second and third compounds are inactive against HIV, as
well as the inactive L-valine 17 and D-valine derivatives 19
that have been considered for these experiments. The assay
has been carried out by dissolving the compounds in acetone-
d6 and trizma buffer (pH=7.6), incubating with the enzyme
To probe the activation of the ACV ProTides to the
monophosphorylated form inside cells, we performed an

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5525
Figure 5. Carboxypeptidase-mediated cleavage of compound 6, monitored by 31P NMR.
Figure 6. Carboxypeptidase-mediated cleavage of compound 17, monitored by 31P NMR.
and recording a blank for each sample before the addition of
the enzyme.
Also, CEM cell extracts have been prepared to examine
the rate of hydrolysis of the antivirally active 9 and 4 and the
inactive 6 derivatives. Whereas 9 and 4 were efficiently
hydrolyzed within a short time period (>95% conversion
of 9 and 65% conversion of 4 within 1 h of incubation), 6
proved entirely stable after a 120 min incubation period
(Figure 8). These observations are in agreement with the
antiviral data and demonstrate that CEM cell-associated
enzymes can efficiently convert methyl and benzyl esters of
the ACV ProTides but not tert-butyl esters. Tonsil extracts
were also found to efficiently hydrolyze 9 and 4, with the
same preference profile of 9 over 4 as found for the CEM cell
extracts (data not shown).
These experiments support the need of activation of ACV
ProTide in order to deliver the ACV monophosphate meta-
bolite. The enzymatic data correlate well with the in vitro
anti-HIV data and may support the role of carboxypeptidase
Y in the ProTide activation in the lymphocyte cell cultures.
Stability Studies of ACV ProTide. Two different stability
studies of compound 9 using human serum and pH 1.0 buffer
have been conducted. In the case of human serum, 9 was
dissolved in DMSO and D2O and human serum was added.
The experiment was conducted at 37 °C and monitored by
31P NMR. In Figure 9 are reported 31P NMR spectra 10 min
after the addition of the serum and after 12 h. For a better
resolution, both original spectra and deconvoluted ones have
In the case of the phenyl benzylalanine compound 9
(Figure 3), the experiment showed a fast hydrolysis of the
starting material (δP=3.65 and 3.60) to the intermediate type
88 (Figure 1) (δP=4.85 and 4.70), noting the presence of the two
diastereoisomers. This intermediate is then processed to a
compound of type 90 (δP = 7.10) through the putative inter-
mediate 89, which is not detected by 31P NMR. The half-life for 9
is 17 min. In the case of the isopropyl ester analogue 10
(Figure 4), the experiment showed a slow conversion of the
starting material to 90 with a half-life of 46 h. This is ca. 150 times
slower than 9. This result is in accordance with the inactivity of
10 against HIV (Table 2). Notably one of the two diastereo-
isomers seems to be faster converted compared to the other one.
Compound 6, the naphthyl t-butyl alanine analogue
(Figure 5), showed no conversion at all presumably due to
the presence of the tert-butyl ester, which is too bulky to be
processed by the enzyme. This observation is in agreement
with the lack of antiviral activity for this compound.
In the case of the L-valine derivative 17 (Figure 6), the
experiment showed, as already demonstrated for compound
10, a slow conversion to the compound of type 90, with a
half-life of 72 h. The D-valine derivative 19 (Figure 7) was not
processed due to the presence of the non-natural amino acid,
which seems not to be recognized by the enzyme.

5526 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Derudas et al.
Figure 7. Carboxypeptidase-mediated cleavage of compound 19, monitored by 31P NMR.
Figure 8. Stability of ACV ProTides in crude CEM cell extracts as a function of incubation time.
Figure 9. Stability of compound 9 in human serum, monitored by 31P NMR.
been reported. The spectra show that the ACV ProTide is
stable under these conditions. In fact, after 12 h, 56% of the
compound is still present. The spectra also show the forma-
tion of the compound type 90 and the formation of a peak at
δP = 1.90, which may correspond to the monophosphate
form. The peak at δP=2.25 corresponds to the human serum
that in the first experiment is overlapping with the peak at at
δP=1.90.
added. The experiment was conducted at 37 °C and moni-
tored by 31P NMR. The experiment showed a good stability
of the compound (see Figure 12 in the Supporting In-
formation) having an half-life of 11 h. Notably, the forma-
tion after 5 h of a peak at δP = -0.25, which should
correspond to the monophosphate form was observed.
Molecular Modeling-1: Carboxypeptidase Y Enzyme.
To better understand the enzymatic results obtained using
carboxypeptidase Y, molecular modeling studies using a
crystal structure of the enzyme have been performed.17 The
In the case of the stability in acid, a pH of 1.0 was used.
Compound 9 was dissolved in MeOD, and the buffer was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5527
Figure 11. (a) Interactions of 90 (L-Ala) with the active site of
human Hint-1. (b) Docking of compound 90 (L-Ala) ACV-MP
phosphoramidate within the catalytic site of human HINT (I)
enzyme.
Figure 10. (a) Docking of compound 9 within the catalytic site of
carboxypeptidase Y enzyme. (b) Docking of compound 6 within the
catalytic site of carboxypeptidase Y enzyme.
putative mechanism of action involves an attack from the
Ser146 to the carbonyl of the ester, which is coordinated with
the NH from Gly52 and Gly53.18
with the Gly52 and Gly53. This result supports the fact that
one of them is faster metabolized, presumably the RP, than
the other one, and this is due to a different binding in the
catalytic site of the enzyme. In the case of the valine
derivatives, none of them showed a suitable pose in the active
site of the enzyme.
The processed compound 9 showed a positive interaction
with the active site of the enzyme for both phosphate
diastereoisomers (shown only the RP diastereoisomer)
(Figure 10a). In particular, the carbonyl moiety is in a
suitable position for the nucleophilic attack from the cata-
lytic Ser146, with the NH from Gly52 and Gly53 correctly
placed to stabilize the tetrahedral intermediate. This result is
in accordance with the enzymatic result for this compound.
In the case of the inactive compound 6, the carbonyl is not
in a favorable position, pointing away from Gly52 and
Gly53, probably due to the presence of the bulky tert-butyl,
which may influence the interaction with enzyme resulting in
a poor activation (reported only the SP-diastereoisomer,
Figure 10b). The docking of compound 10, which showed
a faster hydrolysis of one diastereoisomer compared to the
other one, showed interesting results. In fact, the two dia-
stereoisomers docked in a different way. The R-diastereo-
isomer showed a preferable position for the carbonyl moiety,
while in the case of the S-diastereoisomer the position of
carbonyl group is different and it is not able to coordinate
Molecular Modeling-2: Human Hint Enzyme. As shown in
the enzymatic experiment on 9, the first step of activation
proceeds well and leads to compound 90, which needs to be
further converted in order to release the monophosphate
form 91. The last step of the activation of the ProTide
involving the cleavage of the P-N bond is not well-known,
and it is considered to be mediated by a phosphoramidase-
type enzyme called Hint, belonging to the HIT superfam-
ily.19 A molecular modeling study using human hint enzyme
1, cocrystallized with adenosine monophosphate, has been
performed in order to investigate this last step of activation.
The catalytic activity of this enzyme is due to the presence of
three histidines, which interact with the substrate, and to the
presence of a serine, which binds the nitrogen of the amino
acid, protonating the nitrogen, and favoring P-N bond
cleavage (Figure 11a). From Figure 11b, it is clear to see
how the compound binds correctly in the active site of the

5528 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Derudas et al.
enzyme positioning the phosphate moiety (pink) in a suitable
position for the cleavage of the P-N bond. This experiment
suggests that the last step of the activation to release ACV-
MP may proceed well in the case of ACV alaninyl phosphate
in vivo, supporting the biological data.
solvent was removed under reduced pressure and the residue
triturated with anhydrous diethyl ether. The precipitate was
filtered under nitrogen and the solution was concentrated to give
an oil. Most of the aryl phosphorochloridates synthesized were
purified by flash column chromatography (eluting with ethyl
acetate/petroleum ether in different proportions).
Standard Procedure C: Synthesis of Phosphoramidates (67-
85). To a stirring suspension of N2-DMF-ACV (1.00 mol/equiv)
in anhydrous THF was added dropwise under an argon atmo-
sphere tBuMgCl (2.00 mol/equiv), and the reaction mixture was
stirred at room temperature for 30 min. Then was added
dropwise a solution of the appropriate phosphorochloridate
(1.50 to 4.00 mol/equiv) in anhydrous THF. The reaction
mixture was stirred at room temperature overnight. The solvent
was removed under reduced pressure, and the residue was
purified by column chromatography eluting with DCM/MeOH
in different proportions.
Standard Procedure D: Deprotection of N2-DMF-Phosphor-
amidates (2-23). A solution of 67-87 in 1-propanol or
2-propanol was stirred under reflux for 24-96 h. The solvent
was then removed under reduced pressure, and the residue was
purified by column chromatography eluting with DCM/MeOH
in different proportions. The product was usually further pur-
ified by preparative TLC or semipreparative HPLC to give a
white solid.
Conclusion
A series of 22 acyclovir ProTides has been reported. These
compounds as well as the parent ACV have been biologically
evaluated against HSV-1 and -2 and against HIV-1 and -2.
In the case of HSV-1 and -2, the compounds did not show
any improvement of activity compared to the parent. How-
ever, these compounds retained activity against the TK-
deficient HSV-1 strain while ACV showed a loss on activity.
These results showed a successful thymidine-kinase bypass.
In the case of HIV-1 and -2, ACV did not show any acti-
vity, while the ProTides show a good activity in a range of
6.2-17 μM (HIV-1, CEM), 8.9-42 μM (HIV-2, CEM), and
0.8-30 μM (HIV-1, MT-4).
The variation of the amino acid moiety seems to be
tolerated in the case of HSV. In the case of HIV, this variation
is less tolerated, showing good results only in the case of the L-
alanine derivatives and phenylalanine derivative. In the MT-4
cell cultures, dimethylglycine, and L-leucine are tolerated.
These differences on activity may be due to different substrate
specificities and/or different intracellular levels of enzyme
necessary for the activation of these compounds.
Synthesis of N2-DMF Acyclovir (N0-(9-((2-Hydroxyethoxy)-
methyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformi-
midamide) (66). To a suspension of 1 (1.00 g, 4.44 mmol) in dry
DMF (20 mL) was added N,N-dimethylformamide dimethyl
acetal (2.96 mL, 22.20 mmol) and the reaction mixture was
stirred at room temperature for 1 day. After this period, the
solvent was removed and the residue triturated with diethyl
ether and filtered. The solid was washed with diethyl ether to
give a white solid (97%, 1.20 g). 1H NMR (DMSO, 500 MHz): δ
11.30 (1H, s, NH), 8.58 (1H, s, CHN(CH3)2), 7.94 (1H, s, H-8),
5.45 (2H, s, H-10), 4.65 (1H, t, OH), 3.52-3.49 (4H, m, H-40, H-
50), 3.17, 3.04 (6H, 2s, N(CH3)2).
Experimental Section
General. Anhydrous solvents were bought from Aldrich and
used without further purification. All reactions were carried out
under an argon atmosphere. Reactions were monitored with
analytical TLC on silica gel 60-F254 precoated aluminum plates
and visualized under UV (254 nm) and/or with 31P NMR
spectra. Column chromatography was performed on silica gel
(35-70 μM). Proton (1H), carbon (13C), and phosphorus (31P)
NMR spectra were recorded on a Bruker Avance 500 spectro-
meter at 25 °C. Spectra were autocalibrated to the deuterated
solvent peak and all 13C NMR and 31P NMR were proton-
decoupled. High resolution mass spectra was performed as a
service by Birmingham University using electrospray (ES).
CHN microanalysis were performed as a service by the School
of Pharmacy at the University of London. Purity (g95%) of all
final products was assured by a combination of microanalysis,
and HPLC, with additional characterization in every case by: H,
C, and P NMR, and HRMS.
Standard Procedure A: Synthesis of Dichlorophosphates (26,
27). To a solution of phosphorus oxychloride (1.00 mol/equiv)
and the appropriate substituted phenol or naphthol (1.00 mol/eq)
in anhydrous diethyl ether, stirred under an argon atmosphere,
and added dropwise at -78 °C under an argon atmosphere
anhydrous TEA (1.00 mol/equiv). Following the addition, the
reaction mixture was stirred at -78 °C for 30 min and then at
room temperature overnight. Formation of the desired com-
pound was monitored by 31P NMR. The mixture was filtered
under nitrogen and the corresponding filtrate reduced to dryness
to give the crude product as an oil.
Synthesis of 1-Naphthyl Dichlorophosphate (26). Prepared
according to standard procedure A, using 24 (4.00 g, 27.74
mmol) in anhydrous diethyl ether (60 mL), POCl3 (2.59 mL,
27.74 mmol), and anhydrous TEA (3.87 mL, 27.74 mmol). After
31P NMR, the solvent was removed under reduced pressure and
the residue was triturated with anhydrous diethyl ether. The
precipitate was filtered, and the organic phase was removed
under reduced pressure to give an oil (95%, 6.91 g). 31P NMR
1
(CDCl3, 202 MHz): δ 3.72. H NMR (CDCl3, 500 MHz): δ
8.02-8.00 (1H, m, H-8), 7.81-7.80 (1H, m, H-5), 7.72-7.70
(1H, m, H-4), 7.54-7.45 (4H, m, H-2, H-3, H-6, H-7).
Synthesis of 1-Naphthyl(benzoxy-L-alaninyl)-phosphorochlor-
idate (45). Prepared according to standard procedure B, 26
(6.91 g, 26.48 mmol), L-alanine benzyl ester tosylate 29 (9.30 g,
26.48 mmol), and anhydrous TEA (7.40 mL, 52.96 mmol) in
anhydrous DCM (100 mL). The reaction mixture was stirred at
-78 °C for 1 h, then at room temperature for 2 h. The crude was
purified by column chromatography eluting with ethyl acetate/
hexane=5/5 to give an oil (72%, 7.68 g). 31P NMR (CDCl3, 202
MHz): δ 8.14, 7.88. 1H NMR (CDCl3, 500 MHz): δ 7.99-7.25
(12H, m, Naph, OCH2Ph), 5.15-5.07 (2H, m, CH2Ph), 4.30-
4.23 (1H, m, CHCH3), 1.49-1.46 (3H, m, CHCH3).
Synthesis of N2-DMF-acyclovir-[1-naphthyl(benzoxy-L-alani-
nyl)] Phosphate (67). Prepared according to standard procedure
C, from 66 (0.30 g, 1.07 mmol) in anhydrous THF (10 mL),
tBuMgCl (1.0 M THF solution, 2.14 mL, 2.14 mmol), 45 (1.31 g,
3.25 mmol) in anhydrous THF (10 mL), and the reaction
mixture was stirred at room temperature overnight. The residue
was purified by column chromatography, eluting with DCM/
MeOH=95/5, to give a white solid (17%, 0.12 g). 31P NMR
(MeOD, 202 MHz): δ 4.18, 3.92. 1H NMR (MeOD, 500 MHz):
δ 8.47, 8.46 (1H, 2s, NCHN(CH3)2), 8.01-7.98 (1H, m, H-8
Naph), 7.78-7.74 (2H, m, H-8, H-6 Naph), 7.56, 7.55 (1H, m,
Standard Procedure B: Synthesis of Phosphorochloridates
(45-65). To a stirred solution of the appropriate aryl dichlor-
ophosphate 26-28 (1.00 mol/equiv) and the appropriate amino
acid ester salt 29-44 (1.00 mol/equiv) in anhydrous DCM was
added dropwise at -78 °C under an argon atmosphere, anhy-
drous TEA (2.00 mol/equiv). Following the addition the reac-
tion mixture was stirred at -78 °C for 30 min to 1 h and then at
room temperature for 30 min to 3.5 h. Formation of the desired
compound was monitored by 31P NMR. After this period, the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5529
H-2 Naph), 7.41-7.12 (9H, m, Naph, OCH2Ph), 5.37-5.36
(2H, 2s, H-10), 5.00-4.93 (2H, m, OCH2Ph), 4.14-4.06 (2H, m,
H-50), 3.96-3.88 (1H, m, CHCH3), 3.88-3.59 (2H, m, H-40),
2.95-2.93 (6H, m, N(CH3)2), 1.20-1.17 (3H, m, CHCH3).
Synthesis of Acyclovir-[1-naphthyl(benzoxy-L-alaninyl)] Phos-
phate (2). A solution of 67 (0.10 g, 0.16 mmol) in 2-propanol
(5 mL) was stirred under reflux for 2 days. The solvent was then
removed under reduced pressure, and the residue was purified
by column chromatography eluting with DCM/MeOH=96/4.
The product was purified by preparative TLC (gradient elution
of DCM/MeOH=99/1, then 98/2, then 96/4) to give a white
solid (35%, 0.032 g). 31P NMR (MeOD, 202 MHz): δ 4.13, 3.96.
1H NMR (MeOD, 500 MHz): δ 8.01-7.99 (1H, m, H-8 Naph),
7.77-7.75 (1H, m, H-6 Naph), 7.67, 7.64 (1H, 2s, H-8), 7.58-
7.13 (10H, m, Naph, OCH2Ph), 5.28, 5.25 (2H, 2s, H-10), 4.99-
4.94 (2H, m, OCH2Ph), 4.12-4.06 (2H, m, H-50), 3.97-3.93
(1H, m, CHCH3), 3.64-3.59 (2H, m, H-40), 1.24-1.20 (3H, m,
CHCH3). 13C NMR (MeOD, 125 MHz): δ 20.32 (d, JC-P=7.63,
CHCH3), 20.43 (d, JC-P = 6.61, CHCH3), 51.76, 51.81 (2s,
CHCH3), 67.20 (d, JC-P=5.58, C-50), 67.28 (d, JC-P=4.91, C-
50), 67.95, 67.98 (2s, OCH2Ph), 69.34 (d, JC-P=7.72, C-40), 69.40
(d, JC-P = 8.14, C-40), 73.65 (C-10), 116.26, 116.29, 116.35,
122.69, 122.80, 125.92, 126.51, 127.20, 127.42, 127.46, 127.74,
128.81, 128.83, 129.27, 129.33, 129.52, 129.57 (C-5, C-2 Naph,
C-3 Naph, C-4 Naph, C-5 Naph, C-6 Naph, C-7 Naph, C-8
Naph, C-8a Naph, OCH2Ph), 136.26, 137.23 (C-4a Naph,
“ipso” OCH2Ph), 139.69 (C-8), 147.98, 148.04 (“ipso” Naph,
C-4), 152.44 (C-2), 159.39 (C-6), 174.61, 174.88 (2s,
COOCH2Ph). EI MS=615.1735 (M þ Na). Anal. Calcd for
Data were collected and analyzed using Nucleoview software
(Chemometec, Denmark).
Human tonsils obtained under an IRB-approved protocol
were dissected into ∼2 mm blocks and cultured on collagen rafts
at the medium-air interface. Tissues were inoculated ex vivo
with X4LAI.04 (∼0.5 μg of p24gag per block) and treated with
ACV ProTides at concentrations ranging from 0.1 to 10 μM.
The culture medium was changed every 3 days, and ACV
ProTides were replenished. For each compounds’ concentration
HIV-1 release was quantified by measurements of p24gag accu-
mulated over 3-day periods in the culture media bathing 18
tissue blocks. The EC50 corresponded to the compound con-
centration required to suppress by 50% the production of p24.
Preparation of CEM and Tonsil Cell Extracts and Analysis of
ProTide Conversion. Exponentially growing CEM cells or tonsil
tissues were washed twice with PBS. Then, cells and tissues were
suspended in PBS, and extracts were made in a Precellys-24
homogenizator (Berlin Technologies, Montigny-en-Breton-
neux, France) (tonsils) or by a Hielscher-Ultrasound Technol-
ogy (CEM cells) (Germany). The extracts were cleared by
centrifugation (10 min, 15000 rpm) and frozen at -20 °C before
use. Ten micromolar solutions of 9, 4, and 6 were added to the
crude cell and tissue extracts (100 μL) and incubated for 30, 60,
and 120 min at 37 °C. At each time point, 20 μL of the incubation
mixtures were withdrawn and added to 30 μL cold methanol to
precipitate the proteins. After centrifugation, the supernatants
were subjected to HPLC analysis on a reverse phase C18 column
(Merck) to separate the parent ACV ProTides from their
hydrolysis products that may be formed during the incubation
process. Data were plotted as percent of disappearance of the
intact parent ACV ProTide from the incubation mixture.
C28H29N6O7P 0.5H2O: C, 55.91; H, 5.03; N, 13.97. Found: C,
3
55.81; H, 4.91; N, 13.78.
Antiviral Activity Assays. The compounds were evaluated
against the following viruses: HSV-1 strain KOS, thymidine
kinase-deficient (TK-) HSV-1 KOS strain resistant to ACV
(ACVr), HSV-2 strain G, HIV-1 strain IIIB/Lai, and HIV-2
strain ROD. The antiviral, other than anti-HIV, assays were
based on inhibition of virus-induced cytopathicity or plaque
formation in human embryonic lung (HEL) fibroblasts. Con-
fluent cell cultures in microtiter 96-well plates were inoculated
with 100 CCID50 of virus (1CCID50 being the virus dose
required to infect 50% of the cell cultures). After a 1-2 h
adsorption period, residual virus was removed and the cell
cultures were incubated in the presence of varying concentra-
tions of the test compounds. Viral cytopathicity was recorded as
soon as it reached completion in the control virus-infected cell
cultures that were not treated with the test compounds. Anti-
viral activity was expressed as the EC50 or effective compound
concentration required to reduce virus-induced cytopathicity by
50%.
Acknowledgment. Marco Derudas dedicates this work to
the memory of Antonietta Derudas. We thank Frieda De
Meyer, Leentje Persoons, Vicky Broeckx, Leen Ingels, and
Ria Van Berwaer for excellent technical assistance with the
antiviral and enzymatic assays. We acknowledge the excellent
administrative support of Helen Murphy. Financial support
by a grant of the KU Leuven (GOA no. 05/19) was provided.
The work of A.L., C.V., and L.M. was supported by the
NICHD Intramural Program.
Supporting Information Available: Preparative methods,
spectroscopic and analytical data on target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Human CEM cell cultures (∼3ꢀ105 cells mL-1) were infected
with 100 CCID50 HIV-1(IIIB) or HIV-2(ROD) per mL and
seeded in 200 μL well microtiter plates, containing appropriate
dilutions of the test compounds. After 4 days of incubation at
37 °C, CEM giant cell formation was examined microscopically.
MT-4 cells (1 ꢀ 104 cells per mL) were suspended in fresh
culture medium and infected with 10 μL (0.7 ng of p24) of
X4LAI.04 viral stock per mL of cell suspension. Infected cell
suspensions were then transferred to microplate wells, mixed
with 1 mL of medium containing the test compound at an
appropriate dilution and further incubated at 37 °C. After 3
days, p24 production was measured in the MT-4 cell culture
supernatants. The EC50 corresponded to the compound con-
centration required to suppress the production of p24 in the
virus-infected MT-4 cell cultures by 50%. Viability in MT-4 cell
cultures were evaluated using a nucleocounter automated cell
counting system (Chemometec, Denmark). Total number of
cells and number of dead cells in the cultures untreated and
treated with ACV ProTides were enumerated using a propidium
iodide-based assay according to the manufacturers’ protocol.
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