Successful kinase bypass with new acyclovir phosphoramidate prodrugs

Article abstract of DOI:10.1016/j.bmcl.2008.06.069

Novel phosphoramidates of acyclovir have been prepared and evaluated in vitro against acyclovir-sensitive and -resistant herpes simplex virus (HSV) types 1 and 2 and varicella-zoster virus (VZV). Unlike the parent nucleoside these novel phosphate prodrugs

Full text of DOI:10.1016/j.bmcl.2008.06.069

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4364–4367
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Successful kinase bypass with new acyclovir phosphoramidate prodrugs
a
b
c
Christopher McGuigan a, , Marco Derudas , Joachim J. Bugert , Graciela Andrei ,
*
Robert Snoeck c, Jan Balzarini c
a Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Department of Medical Microbiology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel phosphoramidates of acyclovir have been prepared and evaluated in vitro against acyclovir-sensi-
tive and -resistant herpes simplex virus (HSV) types 1 and 2 and varicella-zoster virus (VZV). Unlike the
parent nucleoside these novel phosphate prodrugs retain antiviral potency versus the ACV-resistant virus
strain, suggesting an efficient bypass of the viral thymidine kinase.
Received 19 May 2008
Revised 18 June 2008
Accepted 19 June 2008
Available online 24 June 2008
Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords:
HSV
Nucleotides
ProTide
Thymidine kinase
Prodrug
Herpes simplex virus infection is often well managed by the use
of acyclovir, (ACV, 1), its prodrug valacyclovir, or related com-
pounds. The widespread use of (1) has led to the emergence of
HSV strains that are resistant to this drug.
recently successfully applied to abacavir for HIV9 and 40-substi-
tuted nucleosides for Hepatitis C Virus (HCV).10
We have previously reported the application of this method to
ACV (1) and the results indicated that the approach failed.11 Thus,
the ProTide (2) derived from (1) was found to be poorly active ver-
Resistance appears uncommon in immunocompetent patients;
Morfin1 reports a prevalence below 1%. A more recent study in
the Netherlands reports a prevalence of 0.27% in this population.2
However, resistance is significantly more common in immunocom-
promised patients. Stranska et al.2 report 7% and Morfin cites 5%.
Notably, the proportion of resistant isolates rises to 30% in patients
receiving allogeneic bone marrow transplants. Three separate
mechanisms of resistance to (1) have been considered to occur; a
loss of viral thymidine kinase (TK) activity, an altered TK substrate
specificity and an alteration of viral DNA polymerase.3 Given the
non-essential nature of the viral TK and the importance of the viral
polymerase, it is unsurprising that the great majority of resistant
isolates correspond to deletion/inactivation of the TK gene.4 One
approach to manage TK-related resistance is to use agents not
requiring HSV TK for activation, such as cidofovir or foscarnet.
However, they may carry a risk of increased toxicity. Another ap-
proach would be to bypass the dependence of (1) on HSV TK by
using a suitable phosphate prodrug, or ProTide. Several such meth-
ods now exist, such as the cyclosal approach,5 ester-based methods
or SATE6 and phosphoramidate diesters.7 Our group has developed
an aryloxy phosphoramidate triester approach,8 which has been
sus HSV2 (EC50 P 100 lM) unlike (1) itself. Compound (2) was
roughly equi-active as (1) versus VZV and slightly more active ver-
sus human cytomegalovirus (HCMV) but there was no clear thera-
peutic advantage. However we have recently reported a new
generation of phosphoramidate protides in which the aryl moiety
is a bicyclic system such as 1-naphthyl.12
We were keen to explore the application of the naphthyl
ProTide methodology to (1) for two reasons. Firstly the observation
that naphthyl for phenyl can give a potency boost and secondly
that these compounds may have a significant lipophilicity enhance-
ment over prior structures. This may be particularly important in
the case of (1) where the inherent lipophilicity is rather low, and
first generation protides may be insufficiently lipophilic for effi-
cient passive diffusion into cells. Indeed, ClogP13 estimates on (2)
indicate a figure of ca. ꢀ0.8; although significantly more lipophilic
than (1) this is somewhat lower than what may be viewed as opti-
mal. Thus, in addition to examining naphthyl phosphates we also
sought more lipophilic esters than the methyl ester (2) previously
reported.
Compounds were prepared from (1) as shown in Scheme 1.
In order to improve the solubility of ACV we protected the
guanine base using N,N-dimethylformamide dimethyl acetal, to
give (7). The coupling with the appropriate phosphorochloridate
* Corresponding author. Tel./fax: +44 2920 874537.
E-mail address: mcguigan@cardiff.ac.uk (C. McGuigan).
0960-894X/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.069

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4364–4367
4365
O
O
N
N
N
N
NH
NH
NH2
N
N
N
N
HO
HO
O
O
(1)
Dimethylacetyl-
formamide
DMF, RT
(7)
ArO(ROC(O)CHR'NH)POCl
tBuMgCl, THF, RT, 17h
O
N
(2-6)
O
N
For Ar, R, R' see table
N
N
N
NH
NH
NH2
N
N
O
N
O
O P O
HN
Ar
O P O
HN
Ar
O
O
R'
2-Propanol
reflux, ca40h
R'
(8)
O
O
O
R
O
R
Scheme 1.
was performed using tert-butyl magnesium chloride as a hydroxyl
activator, to give blocked compounds of type (8).14 The DMF-
protecting group was then removed by refluxing in 2-propanol
(40–72 h). Owing to the chirality of the phosphorus, all of these
compounds have been isolated and tested as a mixture of two dia-
stereoisomers. Their structures have been demonstrated by NMR
Similar data emerge here, with (3) being particularly active and
retaining significant activity in the TKꢀassay. In this case com-
pound (4) shows a similar profile, while (5) is less active. This im-
plies that the ester moiety (benzyl in (3) and (4), versus methyl in
(2) and (5)) is more important than the aryl moiety. Notably, the
amino acid-modified compound (6) which has Phe in place of
Ala, is poorly active in this assay, particularly versus the resistant
virus. This is despite what might be regarded as a near-optimal
lipophilicity for (6) (Table 1) and points to the importance of the
amino acid moiety for activity.
(
31P, 1H and 13C), mass spectroscopy and elemental analysis.15
The target compounds were first evaluated by plaque assay for
their ability to inhibit the replication of ACV-sensitive and
ACV-resistant HSV2 in Vero cells (Table 1).16
As can be seen from Table 1, while the previously reported phe-
nyl methylalanine phosphate (2) is poorly active, being ca 7-fold
less active than (1), in marked contrast to (1) it does retain full po-
tency versus the resistant strain. This implies that (2) does function
as a monophosphate prodrug as intended, but with low efficiency,
particularly in the nucleoside-sensitive assay. By comparison, the
naphthyl phosphate (3) is roughly equi-active with (1) versus
TK-competent virus and notably retains full potency versus
ACV-resistant HSV-2. The simplest explanation of this is that the
ACV-resistant HSV-2 mutant is TK deficient and that (3) is TK-inde-
pendent, strongly implying a successful thymidine kinase bypass.
Notably, ClogP calculations on (3) indicate a significant enhance-
ment over (1) to a figure of ca. 2 which may be regarded as near
optimal. Indeed the ‘mixed’ compounds (4) and (5) have lower
ClogP values and are less active in the HSV-2 TK+ assay, but retain
good activity in the HSV-2 TKꢀ assay. In a further assay in HEL cells
we evaluated the samples against both TK+ and TKꢀ HSV-1 and
HSV-2 with data shown in Table 2.
One concern with bypassing the HSV-TK might be of enhanced
cytotoxicity and loss of antiviral selectivity. However, the MCC
data on this series (Table 2) do not reveal a significant toxicity. If,
as appears likely, the viral TK is being bypassed, there must still
be some element of viral specificity at another stage, most likely
at the polymerase level.
In another assay we examined this family of prodrugs against
kinase-competent and kinase deficient VZV (in HEL cells) with data
shown in Table 3. As noted in this table, unlike ACV (1) several of
the agents retain good potency in the TKꢀ VZV assays, notably
compound (4) which essentially retains full potency. Interestingly
(4) is also non-toxic while (3) does have some toxicity here.
Thus, compound (4) emerges as particularly active in a range of
assays. It retains full activity versus all resistant viral strains, HSV-
1, -2 and VZV, being low or sub-lM in most cases, and non-toxic.
In conclusion, we report the successful application of the Pro-
Tide approach to acyclovir. The naphthyl and phenyl benzyl ala-
nine ProTides are fully active in vitro against ACV—resistant
Table 1
Anti-HSV-2 activity of ProTides
a
Compound
Ar
R
Amino acid
ClogP
EC50
(l
M)
HSV2-HG32 (ECACC 158)
HSV2-ACVR (ECACC 513)
1
2
3
4
5
6
—
Ph
1-Nap
Ph
1-Nap
Ph
—
—
ꢀ2.42
ꢀ0.82
2.06
0.89
0.35
6 1.3
43.1 10.6
9.8 2.2
20.3 6.6
40.4 13.4
20.3 6.6
>100
Me
Bn
Bn
Me
Bn
Ala
Ala
Ala
Ala
Phe
18.7 7.2
14.5 6.6
15.5 7.4
15 6.2
2.31
33.2 3.9
a
Values are means of three experiments, with standard deviations given, in Vero cells.

4366
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4364–4367
Table 2
Anti-HSV-1 and -2 activity of ProTides
a
b
Compound
Ar
R
Amino acid
EC50
/
l
M
MCC
(lM)
HSV-1 (Kos)
HSV-2 (G)
HSV-1 TK-Kos ACVR
1
2
3
4
5
6
—
Ph
1-Nap
Ph
1-Nap
Ph
—
—
0.4
20
2
0.9
16
17
0.2
15
1.4
1.4
10.4
8
50
80
11
8
80
>100
>250
>100
>100
>100
>100
>100
Me
Bn
Bn
Me
Bn
Ala
Ala
Ala
Ala
Phe
a
EC50, 50% effective concentration that inhibits virus-induced cytopathicity by 50%, in HEL cells.
MCC, minimal cytotoxic concentration that causes a microscopically visible alteration of cell morphology.
b
Table 3
Anti-VZV activity of ProTides
MCCb
(lM)
CC50 (lM)
a
c
Compound
EC50
(l
M)
OKA TK+
YS TK+
07/1TKꢀ
YS/RTKꢀ
1
2
3
4
5
6
2.5
19
7.2
0.72
7.6
5.2
2.9
20
3.3
1.0
10.9
6.6
61
24
6.9
1.8
22
43
16
ND
0.59
6.1
10.0
>500
>100
>50
>50
>50
1350
162
20
>100
>100
86.9
8.4
>50
a
EC50, 50% effective concentration that inhibits virus-induced cytopathicity by 50%, in HEL cells.
MCC, minimal cytotoxic concentration that causes a microscopically visible alteration of cell morphology.
CC50, 50% cytostatic concentration that inhibits cell proliferation by 50%.
b
c
8. Cahard, D.; McGuigan, C.; Balzarini, J. MiniRev. Med. Chem. 2004, 4, 371.
viruses and appear to represent successful thymidine kinase by-
pass. Notably the ProTides do not in general suffer from significant
cytotoxicity.
9. McGuigan, C.; Harris, S. A.; Daluge, S. M.; Gudmundsson, K. S.; McLean, Ed. W.;
Burnette, T. C.; Marr, H.; Hazen, R.; Condreay, L. D.; Johnson, L.; Clercq, E.; De
Balzarini, J. J. Med. Chem. 2005, 48, 3504.
10. Perrone, P.; Luoni, G. M.; Kelleher, M.-R.; Daverio, F.; Angell, A.; Mulready, S.;
Congiatu, C.; Rajyaguru, S.; Martin, J. A.; Léêue, V.; Le Pogam, S.; Najera, I.;
Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med. Chem. 2007, 50, 1840.
11. McGuigan, C.; Slater, M. J.; Parry, N. R.; Perry, A.; Harris, S. BioOrg. Med. Chem.
Lett. 2000, 10, 645.
12. Congiatu, C.; McGuigan, C.; Jiang, W. G.; Davies, G.; Mason, M. D. Nucleosides
Nucleotides Nucleic Acids 2005, 24, 485.
13. ClogP calculations based on ChemDraw Ultra 9.0.
The phenyl benzyl alanine compound (4) appears a particularly
promising lead for further development.
These data strongly support the notion that ProTide derivatives
are successful in the intracellular delivery of the monophosphate of
ACV and bypass the dependence of the nucleoside analogue on
(viral) thymidine kinase. Interestingly, the naphthyl phosphate is
not a pre-requisite for activity, and some phenyl phosphates dis-
play good potency, particularly in kinase-deficient cells. Modifica-
tion of the ester moiety or the aryl, or both, seems to be beneficial
to tune the ProTide for optimal activity. Although the overall lipo-
philicity may be an important feature for activity, it is not the only
determinant.
14. Uchiyama, M.; Aso, Y.; Noyori, R.; Hayakawa, Y. J. Org. Chem. 1993, 58, 373.
15. Procedures for the preparation of (3): Synthesis of N2-DMFacyclovir-[1-
naphthyl(benzoxy-
L
-alaninyl)]phosphate (8). To
a
stirring suspension/
solution of N2-DMF acyclovir (7) (0.30 g, 1.08 mmol) in anhydrous THF
(10 mL) was added, tBuMgCl (1.0 M THF solution, 2.16 mL, 2.16 mmol),
dropwise under an argon atmosphere. After 30 min, 1-naphthyl(benzoxy-
L
-alaninyl)-phosphorochloridate (1.31 g, 3.25 mmol, 2.00 mol/eq) was added
dropwise in dry THF (10 ml) and 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/(95:5),
to give a colourless solid (17%, 0.12 g).31P NMR (MeOD, 202 MHz): d 4.18,
3.92.1H NMR (MeOD, 500 MHz): d 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, 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-40), 3.96–3.88 (1H, m, CHCH3), 3.88–3.59
(2H, m, H-30), 2.95–2.93 (6H, m, N(CH3)2), 1.20–1.17 (3H, m, CHCH3). Synthesis
We are exploring further the limits of the technology on acyclic
nucleoside analogues and the opportunities that by-passing the
viral kinase may bring.
Acknowledgments
We thank the Sardinian ‘Master and Back’ Programme for fund-
ing and the Geconcerteerde Onderzoeksacties (GOA No. 05/15) also
Helen Murphy for excellent secretarial assistance. We thank Mrs.
Lies Van den Heurck, Mrs. Anita Camps, Mr. Steven Carmans,
Mrs. Frieda De Meyer, Mrs. Vicky Broeckx, Mrs. Leentje Persoons
and Mr. Keith Williams for excellent technical assistance.
of acyclovir-[1-naphthyl(benzoxy-L-alaninyl)]phosphate (3). A solution of the
protected protide (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 colourless solid (35%,
0.032 g). 31P NMR (MeOD, 202 MHz): d 4.13, 3.96. 1H NMR (MeOD, 500 MHz): d
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-40), 3.97–3.93 (1H, m, CHCH3), 3.64–3.59
(2H, m, H-30), 1.24–1.20 (3H, m, CHCH3). 13C NMR (MeOD, 125 MHz): d 20.32
(d, CH3, JC–P = 7.63) 20.43 (d, CH3, JC–P = 6.61), 51.76, 51.81 (2s, CHCH3), 67.20
(d, C-40, JC–P = 5.58), 67.28 (d, C-40, JC–P = 4.91) 67.95, 67.98 (2s, OCH2Ph), 69.34
(d, C-30, JC–P = 7.72), 69.40 (d, C-30, JC–P = 8.14),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 (COOCH2Ph). EI MS = 615.17 (M+Na).
16. Biological methods: Table 1. Vero cells (ECACC #84113001) were maintained
in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine
References and notes
1. Morfin, F.; Thouvenot, D. J. Clin. Virol. 2003, 26, 29.
2. Stranska, R.; Schuurman, R.; Nienhuis, E.; Goedegebuure, I. W.; Polman, M.;
Weel, J. F.; Wertheim-Van Dillen, P. M.; Berkhout, R. J. M.; VanLoon, A. M. J. Clin.
Virol. 2005, 32, 7.
3. Larder, B. A.; Cheng, Y.-C. ; Darby, G. J. Gen. Virol. 1983, 64, 523.
4. Nugier, F.; Colin, J. N.; Aymard, M.; Larylois, M. J. Med. Virol. 1992, 36, 2.
5. Meier, C.; Balzarini, J. Antiviral Res. 2006, 71, 282.
6. Peyrottes, S.; Egron, D.; Lefebure, I.; Gosselin, G.; Imbach, J. L. MiniRev. Med.
Chem. 2004, 4, 395.
7. Drontle, D. P.; Wagner, C. R. MiniRev. Med. Chem. 2004, 4, 409.

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4364–4367
-glutamine, 105 IU/ml penicillin and 100 g/ml
4367
serum, 300
l
g/ml
L
l
[herpes simplex virus type 1 (HSV-1) (KOS and KOS-R) and herpes simplex
virus type 2 (HSV-2) (G)]. Confluent HEL cell cultures in 96-well microtiter
plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose
to infect 50% of the cell cultures). After 1-hour virus adsorption period, residual
virus was removed, and the cells were incubated in the presence of serial
dilutions of the test compounds. Viral cytopathicity was recorded within 48 h.
For varicella-zoster virus [VZV (wild-type Oka and YS and TKꢀ deficient 07/1
and YS-R)], the inhibition of plaque formation was recorded. Confluent HEL
cells in 96-well microtiter plates were infected with 20 plaque forming units
(PFU)/ well. After 2-hours incubation, residual virus was removed and the cells
were incubated in the presence of the compounds. Virus plaque formation was
streptomycin and grown in 24-well plates to density. Appropriate wells were
preincubated for 30 minutes with drug (prediluted in DMEM without
additives), and kept growing in the appropriate amount of drug over the
course of the experiment. HSV 2 strain (100 pfu) HG32 (ECACC # 158) and HSV
2 ACR (ECACC # 513) were inoculated per well, adsorbed for 45 min and then
overlayed with 1.2% Avicel RC-591 (Camida Ltd.) suspended in DMEM. After 3
days the overlay was removed and the cells stained with crystal violet, the
plates were scanned and the plaques counted. All assays were run in
quadruplicate on each plate and plaques were counted as averages of four
assays. EC50 was expressed as averages with standard deviation of
3
experiments.Tables 2 and 3. The antiviral assays were based on inhibition of
virus-induced cytopathicity in human embryonic lung (HEL) fibroblasts
recorded after 5 days.17
17. Matrosovich, M.; Matrosovich, T.; Garten, W.; Klenk, H. D. Virol. J. 2006, 3, 63.
.