Synthesis, transport and antiviral activity of Ala-Ser and Val-Ser prodrugs of cidofovir

Article abstract of DOI:10.1016/j.bmcl.2011.04.126

We report the synthesis and biological evaluation of Ala-(Val-)l-Ser- CO₂R prodrugs of 1, where a dipeptide promoiety is conjugated to the P(OH)₂ group of cidofovir (1) via esterification by the Ser side chain hydroxyl group and an e

Full text of DOI:10.1016/j.bmcl.2011.04.126

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4045–4049
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, transport and antiviral activity of Ala–Ser and Val–Ser prodrugs
of cidofovir
Larryn W. Peterson a, Jae-Seung Kim b, Paul Kijek b, Stefanie Mitchell b, John Hilfinger b, Julie Breitenbach c,
Kathy Borysko c, John C. Drach c,d, Boris A. Kashemirov a, Charles E. McKenna a,
⇑
a Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA
b TSRL Inc., Ann Arbor, MI 48108, USA
c College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065, USA
d School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and biological evaluation of Ala–(Val–)
L
-Ser–CO2R prodrugs of 1, where a dipep-
Received 21 March 2011
Accepted 26 April 2011
Available online 3 May 2011
tide promoiety is conjugated to the P(OH)2 group of cidofovir (1) via esterification by the Ser side chain
hydroxyl group and an ethyl group (4 and 5) or alone (6 and 7). In a murine model, oral administration of
4 or 5 did not significantly increase total cidofovir species in the plasma compared to 1 or 2, but 7
resulted in a 15-fold increase in a rat model and had an in vitro EC50 value against human cytomegalo-
Keywords:
Prodrug
Cidofovir
virus comparable to 1. Neither 6 nor 7 exhibited toxicity up to 100 lM in KB or HFF cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Antiviral
Human cytomegalovirus
Oral bioavailability
Cidofovir (HPMPC, 1) is a broad-spectrum antiviral agent
(VistideÒ) used to treat AIDS-related human cytomegalovirus
(HCMV) retinitis.1,2 Currently, cidofovir is of particular interest as
been applied to cidofovir and analogous prodrugs have been
described.17,18
We report here the synthesis and biological evaluation of two
novel classes of cidofovir prodrugs in which a peptidomimetic pro-
moiety is attached to the phosphonate via a serine side chain hy-
droxyl group. In one version, both charges on the phosphonate
a
potential therapy for orthopox virus infections, including
smallpox.3 An important limitation of cidofovir as a therapeutic
agent is its low oral bioavailability (less than 5%)4,5 and poor
transport into cells, which is attributed to its ionizable phosphonic
diacid group. This problem necessitates intravenous administra-
tion, and therefore the development of an orally available form
of cidofovir would be highly advantageous. As a result, there are
efforts to increase the bioavailability of 1 by attachment of auxilia-
ries to the parent drug to mask the anionic phosphonate group.
Several prodrug strategies have been considered to circumvent
this problem.6–9 We previously reported the synthesis and biolog-
ical evaluation of a series of peptidomimetic cyclic cidofovir pro-
drugs containing an amino acid or dipeptide (3) promoiety linked
through an amino acid side chain hydroxyl group,10–15 but applica-
tion of this prodrug approach to acyclic nucleoside phosphonates
(ANP), such as 1 itself, was not explored.
are masked: the dipeptide promoiety (X-
one P–OH, while the other is esterified by a simple alkyl (ethyl)
group (X = -Ala, R = Me (4) or X = -Val, R = Me (5) (Fig. 1)). In
the other, one P–OH is left unmodified, where X = -Val, R = iPr
(6) or X = -Val, R = iPr (7) (Fig. 1).
Mono-esterification of using PyBOP coupling conditions
L-Ser–CO2R) esterifies
L
L
L
D
1
proved difficult as intramolecular cyclization intervened. However,
4 and 5 could be synthesized starting from the trityl-protected
monoethyl ester salts 10 or 11, which in turn can be prepared from
a readily available intermediate (8) from the synthesis of 1.13 Be-
cause 8 is a diethyl ester, it is soluble in organic solvents, which
facilitates purification. Synthesis of 8 was carried out as previously
described,13,19 and the crude product was purified by centrifugal
chromatography on silica gel using a gradient of dichloromethane
and acetone.
Alternative prodrug strategies6,8,16 involving esterification of
the drug phosphonic acid groups with an ether lipid alcohol have
Phosphate triesters or phosphonate diesters can be mono-deal-
kylated by treatment with sodium iodide (NaI)20 or lithium bro-
mide (LiBr)21,22 depending on the alkyl group. Since the reaction
proceeds via an SN2 attack of the halide on the alkyl group, NaI is
commonly used for mono-dealkylation of methyl esters, whereas
⇑
Corresponding author. Tel.: +1 213 740 7007; fax: +1 213 740 0930.
E-mail address: mckenna@usc.edu (C.E. McKenna).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.126

4046
L. W. Peterson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4045–4049
NH2
NH2
N
NH2
N
NH2
N
NH2
N
N
N
O
N
N
N
O
O
P
OH
O
O
O
O
N
O
P
OEt
O
O
O
O
O
P
OH
O
O
O
O
O
O
O
P
O
O
O
O
O
O
O
O
HN
OH
HN
R
OH
O
OH
HN
OH
P
OH
NH2
NH2
NH2
1
2
3a
3b
4
5
6
7
L-Val
D-Val
R = CH3
R = CH(CH3)2
L-Val
D-Val
Figure 1. Structures of cidofovir (1), cyclic cidofovir (2), cyclic cidofovir prodrugs (3), and the prodrugs of 1 synthesized: X-
-Val (5), and X- -Ser–CO2iPr HPMPC, where X = -Val (6) and -Val (7).
L-Ser–CO2Me HPMPC(OEt), where X = L-Ala (4) and
L
L
L
D
LiBr is preferred for more sterically-hindered esters, such as ethyl
esters.23 The reaction does not proceed beyond the formation of
the phosphate diester (or the phosphonate monoester) because at-
tack by the anion on the alkyl group is prevented by the presence
of the negative charge on the monosodium or monolithium salt.20
The monoester 10 was therefore prepared as shown in
Scheme 1. Purified 8 and LiBr were dissolved in dry acetonitrile,
and the solution was refluxed for 30 h. The reaction was monitored
by 31P NMR. Additional equivalents of LiBr were added, and the
reaction was continued until completed.
through a column containing Dowex 50WX8 resin, followed by pre-
cipitation of pure 11 with diethyl ether (73% yield). Besides much
improved yields, this method provides a shorter reaction time
and a less complicated product mixture, facilitating purification.
For conjugation of the trityl-protected cidofovir monoethyl es-
ters 10 or 11 (although 4 was made from 10, 11 is generally pre-
ferred) with a dipeptide X-Ser promoiety, L-valine and L-alanine
were chosen as the N-terminal amino acids (‘X’) by analogy with
the cyclic cidofovir Val–Ser–CO2R prodrugs (3) previously synthe-
sized and studied in our laboratory.13,14 The Boc-protected dipep-
The 31P NMR spectrum of the reaction product obtained by pre-
cipitation with diethyl ether revealed four peaks. The major peak at
d 15.5 ppm corresponded to the desired product 9. Additional sig-
nals at d 16.0, 21.8, and 22.2 ppm were assigned to the monoester
without the trityl group, the diethyl ester (8), and the diethyl ester
without the trityl group, respectively. When the reaction was re-
peated with varied equivalents of LiBr, reaction times and solvents,
partial loss of the trityl group always persisted.
In view of this problem and the resultant low yields, a better pro-
cedure was sought. Racha et al. described the use of NaOH to selec-
tively remove one of the ethyl esters from a diethyl phosphonate.24
Although mono-dealkylation might be accomplished by treatment
of the diester with BTMS,25 it was found that treatment with NaOH
resulted directly in pure product.24 Treatment of 8 with 1.7 M NaOH
in 1:1 ethanol:water (Scheme 1) mono-dealkylated the phospho-
nate diester, and also cleanly removed the benzoyl group in a one
pot reaction. After stirring at room temperature overnight, the ben-
zoic acid side product was removed by passing the reaction mixture
tide promoieties (12) were prepared using a standard EDC
coupling method.26 Conjugates 4 and 5 were synthesized as out-
lined in Scheme 2. The Boc-protected intermediate 13 was prepared
by treatment of 10 or 11 with the N-Boc-protected dipeptide pro-
moiety (12) using PyBOP and DIPEA in DMF at 40 °C. The reaction
was easily monitored by 31P NMR, and when no cidofovir monoeth-
yl ester (10 or 11) remained, the reaction was stopped. The PyBOP
byproduct and excess promoiety were removed with a diethyl ether
wash before aqueous workup and purification of crude 13 by pre-
parative silica gel TLC. Following deprotection of the Boc and trityl
groups with TFA in CH2Cl2, the target compounds 4 and 5 were puri-
fied by preparative TLC and isolated by precipitation with diethyl
ether from a saturated methanolic solution (34% and 7%, respec-
tively). Their structures were confirmed by 1H and 31P NMR and
by HR-MS.27 For conjugates 4 and 5, a chiral center at phosphorus
is formed when the promoiety is attached. Consequently, 4 and 5
were isolated as the resulting diastereomeric mixtures, the compo-
sition of which was determined by 31P NMR.
O
O
NH2
HN
Ph
HN
Ph
O
N
N
N
a
b
O
N
O
P
O
N
O
P
OEt
O
N
O
P
OEt
O
O
NH4
O
OEt
O Li
OEt
OTr
OTr
OTr
8
9
10
O
NH2
N
HN
Ph
N
N
c
O
O
P
O
N
O
P
OEt
O
OH
O
OEt
OEt
OTr
OTr
8
11
Scheme 1. Preparation of 10 and 11. Reagents and conditions: (a) LiBr, CH3CN, reflux, 40 h, 50%; (b) NH4OH, CH3OH, rt, 16 h, 62%; (c) 1.7 M NaOH, H2O/EtOH, rt, 16 h; Dowex
50WX8; Et2O, 73%.

L. W. Peterson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4045–4049
4047
NH2
NH2
N
N
N
OH
O
N
O
P
OEt
O
O
H
N
a
+
O
O
P
OEt
O
O
O
O
O
Boc
N
H
O
OX
HN
R
R
O
OTr
O
OTr
N
H
O
10 or 11
12
13
NH2
N
N
O
O
P
OEt
O
b
13
O
O
O
O
HN
R
OH
NH2
4 R = CH3
5 R = CH(CH3)2
Scheme 2. Synthesis of dipeptide HPMPC(OEt) conjugates 4 and 5. Reagents and conditions: (a) 10 (X = NH4) or 11 (X = H), PyBOP, DIPEA, DMF, 40 °C, 5 h; (b) TFA, CH2Cl2, rt,
16 h. Overall yields: 4, 34%; 5, 7%.
A synthetic route previously reported for preparation of alkox-
yalkyl esters of cidofovir involved coupling 2 with an alkoxyalkyl
alcohol using Mitsunobu chemistry.28 The phosphonate ring was
then opened by heating the product in 2 M NaOH to 80 °C, produc-
ing an acyclic cidofovir derivative in yields of 32–85%.28 These
relatively harsh conditions, if applied to synthesis of a peptidomi-
metic cyclic cidofovir conjugate, such as 13 (Scheme 2) would
likely result in decomposition due to attack of OHÀ on the methyl
ester.
Replacement of the carboxylate methyl ester with an isopropyl
ester10 (14 and 15; Scheme 3) was also desirable to increase the
stability of the dipeptide moiety against enzymatic degradation.
Incorporation of the isopropyl ester presumably stabilized the
promoiety against removal by beta-elimination10 and allowed
the isolation of the first acyclic analogue of this class. To further
suppress decomposition of the prodrug by cleavage of the exocy-
clic rather than the endocyclic P-O bond, milder conditions for
opening the phosphonate ring were explored. When 14 and 15
were placed in bicarbonate buffer at pH 7.5, the phosphonate ring
was opened in good yield to produce the acyclic monoester, Val–
Ser–CO2iPr HPMPC (6 and 7; Scheme 3). Because the formation of
some cyclic cidofovir (2) could not be avoided, 6 and 7 were puri-
fied by preparative HPLC on a C18 column, and then characterized
by 1H and 31P NMR and by HR-MS. Their purity was verified by
HPLC.29
of the drugs into the gastrointestinal tract of a rat at a level of
10 mg/kg (Fig. 2). Oral uptake of 4 was only modestly enhanced,
and 5 showed no significant enhancement compared to 1 and 2.
Furthermore, the only species detected in the plasma was the
monoethyl ester of cidofovir (16; Fig. 3).
When the dipeptide HPMPC(OEt) conjugates 4 and 5 were eval-
uated for oral transport in a murine model, the major species ob-
served in the plasma after oral administration of the prodrug was
again HPMPC(OEt) (16; Fig. 3). Cidofovir monoethyl ester (16)
was therefore evaluated in an in vitro antiviral assay to determine
if: (a) it was processed by cellular enzymes and phosphorylated to
an active triphosphate form; or (b) its ethyl group was removed,
resulting in 1. The in vitro antiviral activity of 16 against vaccinia
virus (VV) was examined in a plaque reduction assay using Ara-A
and 1 as positive controls (Fig. 4). No effect on the virus was de-
tected at any concentration of 16 tested (1–100 lM), showing that
16 was inactive as such and was not significantly converted to 1
under the assay conditions.
D-Val-L-Ser–CO2iPr cHPMPC and D-Val-L-Ser–CO2iPr HPMPC (15
and 7) were also evaluated for oral bioavailability by direct injec-
tion of the drug into the gastrointestinal tract of a rat at a level
of 10 mg/kg (Fig. 5). Both prodrugs exhibited enhanced oral bio-
availability relative to 1 and 2. Intriguingly, despite the presence
of an unmodified P–OH group, the acyclic cidofovir analogue 7
showed the largest AUC and Cmax among all the prodrugs tested.
The enhanced bioavailability of these compounds may be partially
due to their enhanced stabilities.
The X-L-Ser–CO2Me HPMPC(OEt) prodrugs 4 (X = L-Ala) and 5
(X =
L-Val) were evaluated for oral availability by direct injection
NH2
N
NH2
N
N
O
O
N
O
a
O
O
H
N
O
P
OH
H
N
O
O
O
NH2
P
NH2
O
OH
O
O
O
O
O
14 L-Val
15 D-Val
6 L-Val
7 D-Val
Scheme 3. Synthesis of the acyclic cidofovir prodrugs 6 and 7. Reagents and conditions: (a) H2O, pH 7.5, rt, 18 h. Yields: 6, 31%; 7, 33%.

4048
L. W. Peterson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4045–4049
500
400
300
200
100
0
1800
1
1
2
3a
4
5
2
3b
1500
15
7
1200
900
600
300
0
0
1
2
3
4
5
Time (h)
0
2
4
6
8
Time (h)
Figure 5. Dose-corrected total cidofovir found in plasma after intestinal dosing of 1,
2, 3b, 15 and 7. Rats were dosed by direct injection of 3 mg (ꢀ10 mg/kg) of drug into
the gastrointestinal tract, and plasma was sampled over a time course of 4 h.
Figure 2. Dose-corrected total cidofovir found in the plasma after oral dosing of
rats with and
by direct injection of 3 mg (ꢀ10 mg/kg) of drug into the
gastrointestinal tract. Plasma was sampled over a time course of 4 h.
4
5
Table 1
Antiviral activity of cidofovir, cyclic cidofovir, and select peptidomimetic prodrugs
Intracellular
IC50
CPX
(lM)
or
NH2
N
in vivo
VV
HCMV
NH2
N
N
HPMPC (1)
cHPMPC (2)
20
30
0.28, 0.11
0.25
N
40
30
O
O
P
OEt
R
100a
>30a
0.30a
O
O
P
OEt
H
N
L
,
,
L
-CO2Me cHPMPC (3a)
-CO2iPr HPMPC (6)
O
O
O
NH2
>100
>100
—
>100
>100
—
0.32
1.3
3a
L
L
OH
O
D,L
-CO2iPr HPMPC (7)
OH
O
O
OH
Ganciclovir
16
IC50 values for 3a and ganciclovir from Eriksson et al.13
a
4
R = CH3
5 R = CH(CH3)2
Figure 3. Conversion of the dipeptide HPMPC(OEt) conjugates 4 and 5 to 16.
activity against HCMV (IC50 = 0.32 lM), 10-fold lower than ganci-
clovir, the positive control. When the prodrugs were evaluated
for cytotoxicity with KB and HFF cells, no toxicity (CC50) at concen-
125
trations up to 100 lM was observed (Table 2).
The parent compound 1 and its cyclic analogue 2 had in vitro
antiviral activity against the orthopox viruses consistent with pub-
lished values.30 and were more active against HCMV than the pox-
viruses, as were the prodrugs (Table 1). The species-variable
antiviral activity may be due to such factors as different binding
by the target polymerase, or incomplete biotransformation in the
in vitro assays, reflecting differences in the assays themselves. In
particular, the HCMV assay requires incubation for 10 days, but
for the VV and CPX assays, the incubation time is ꢀ3 days. It is pos-
sible that the prodrugs are less potent in the poxvirus assays be-
cause the shorter incubation time did not allow sufficient time
for the prodrug to exert a therapeutic effect by conversion to 1.
Ara-A
100
16
1
75
50
25
0
1
10
100
Drug conc. (µM)
Figure 4. Vaccinia virus plaque reduction assay examining the antiviral activity of
Table 2
16. Ara-A and cidofovir (1), the controls, have EC50 values of
2 and 20 lM,
Cytotoxicity in KB and HFF cells
respectively.
CC50 Values (lM)
KB
HFF
HPMPC (1)
cHPMPC (2)
>100
>100
>100a
>100
100
Prodrugs 6 and 7 were evaluated for in vitro antiviral activity
against VV, cowpox virus (CPX), and human cytomegalovirus
>100a
L
,
,
L
-CO2Me cHPMPC (3a)
-CO2iPr HPMPC (6)
(HCMV). Their EC50 values for VV (>100
lM) were higher than 1
>100
>100
>100
>100
L
L
and 2 (20–40 M; Table 1). A similar pattern was observed for
l
D,L
-CO2iPr HPMPC (7)
CPX with 1 and 2 showing greater activity against this virus than
the prodrugs tested. However, 6 had submicromolar antiviral
CC50 values for 3a from Eriksson et al.13
a

L. W. Peterson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4045–4049
8. Hostetler, K. Y. Antiviral Res. 2009, 82, A84.
4049
Another reason for the observed differences in antiviral activity
might be the expression of different viral-encoded enzymes that
activate the prodrugs.31–35 Recent reports describe the presence
of a serine/threonine phosphatase encoded by HCMV,31,32 while a
dual specificity tyrosine/serine phosphatase, encoded by VV33–35
is expressed late in the virus life cycle.33,35 It is unknown whether
these enzymes would accept the phosphonate-amino acid conju-
gates, and additional studies will be necessary to elucidate the fac-
tors contributing to the observed differences in prodrug antiviral
activity.
9. McGuigan, C.; Gilles, A.; Madela, K.; Aljarah, M.; Holl, S.; Jones, S.; Vernachio, J.;
Hutchins, J.; Ames, B.; Bryant, K. D.; Gorovits, E.; Ganguly, B.; Hunley, D.; Hall,
A.; Kolykhalov, A.; Liu, Y. L.; Muhammad, J.; Raja, N.; Walters, R.; Wang, J.;
Chamberlain, S.; Henson, G. J. Med. Chem. 2010, 53, 4949.
10. McKenna, C. E.; Peterson, L. W.; Kashemirov, B. A.; Serpi, M.; Mitchell, S.; Kim, J.
S.; Hilfinger, J. M.; Drach, J. C. Antiviral Res. 2009, 82, A75.
11. Peterson, L. W.; Kashemirov, B. A.; Blazewska, K. M.; Breitenback, J.; Borysko,
K.; Drach, J. C.; Kim, J. S.; Kijek, P.; Hilfinger, J. M.; McKenna, C. E. Antiviral Res.
2007, 74, A33.
12. Eriksson, U.; Hilfinger, J. M.; Kim, J.-S.; Mitchell, S.; Kijek, P.; Borysko, K. Z.;
Breitenbach, J. M.; Drach, J. C.; Kashemirov, B. A.; McKenna, C. E. Bioorg. Med.
Chem. Lett. 2007, 17, 583.
13. Eriksson, U.; Peterson, L. W.; Kashemirov, B. A.; Hilfinger, J. M.; Drach, J. C.;
Borysko, K. Z.; Breitenbach, J. M.; Kim, J. S.; Mitchell, S.; Kijek, P.; McKenna, C. E.
Mol. Pharm. 2008, 5, 598.
In summary, two classes of peptidomimetic prodrugs of 1 in
which the P(OH)2 was esterified by an Ala–(Val–)L-Ser–CO2R
dipeptide promoiety, with the second P–OH either esterified by
an ethyl group or left unmodified, were synthesized and evaluated
for their transport properties and for their in vitro antiviral activity.
The synthesis of the POEt prodrugs 4 and 5 of 1 was accomplished
using a one pot method to prepare the ethyl ester of trityl-
protected 1 followed by conjugation to the dipeptide promoiety
using PyBOP. After unsatisfactory attempts to mono-esterify the
phosphonate group of 1, prodrugs 6 and 7 were prepared from
their corresponding cyclic forms using basic conditions to open
the phosphonate ring. Administration of 7 to the rat by oral injec-
tion resulted in a 15-fold increase in total cidofovir species in the
plasma relative to 1 or 2, despite the presence of one ionizable
P–OH group in this prodrug. This enhanced oral bioavailability
may be due to higher chemical and enzymatic stability compared
to its cyclic analogue 15. Improved stability might explain why
14. McKenna, C. E.; Kashemirov, B. A.; Eriksson, U.; Amidon, G. L.; Kish, P. E.;
Mitchell, S.; Kim, J.-S.; Hilfinger, J. M. J. Organomet. Chem. 2005, 690, 2673.
15. Peterson, L. W.; Sala-Rabanal, M.; Krylov, I. S.; Serpi, M.; Kashemirov, B. A.;
McKenna, C. E. Mol. Pharm. 2010, 7, 2349.
16. Hostetler, K. Y. Adv. Antiviral Drug Des. 2007, 5, 167.
17. Hostetler, K. Y.; Beadle, J. R.; Hornbuckle, W. E.; Bellezza, C. A.; Tochkov, I. A.;
Cote, P. J.; Gerin, J. L.; Korba, B. E.; Tennant, B. C. Antimicrob. Agents Chemother.
2000, 44, 1964.
18. Hostetler, K. Y.; Rybak, R. J.; Beadle, J. R.; Gardner, M. F.; Aldern, K. A.; Wright, K.
N.; Kern, E. R. Antiviral Chem. Chemother. 2001, 12, 61.
19. Peterson, L. W.; Kashemirov, B. A.; Eriksson, U.; Kim, J. S.; Mitchell, S.; Kijek, P.;
Lee, K. D.; Hilfinger, J. M.; McKenna, C. E. Antiviral Res. 2008, 78, A46.
20. Zervas, L.; Dilaris, I. J. Am. Chem. Soc. 1955, 77, 5354.
21. Krawczyk, H. Synth. Commun. 1997, 27, 3151.
22. Rensing, S.; Arendt, M.; Springer, A.; Grawe, T.; Schrader, T. J. Org. Chem. 2001,
66, 5814.
23. McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M. In Science of Synthesis,
Houben-Weyl Methods of Molecular Transformation; Trost, B. M., Ed.; Georg
Thieme Verlag: Stuttgart, 2009; 42, p 779.
24. Racha, S.; Vargeese, C.; Vemishetti, P.; ElSubbagh, H. I.; Abushanab, E.; Panzica,
R. P. J. Med. Chem. 1996, 39, 1130.
the IC50 value of 7 (1.3 lM) against HCMV in vitro was 4-fold less
than that of 6. We are currently investigating the relationship
between transport and activation, in an attempt to determine an
optimal balance between the two processes.
25. McKenna, C. E.; Schmidhauser, J. J. Chem. Soc., Chem. Commun. 1979, 739.
26. Mahmoud, K. A.; Long, Y. T.; Schatte, G.; Kraatz, H. B. Eur. J. Inorg. Chem. 2005,
173.
27. Compound 4: 1H NMR (400 MHz, CD3OD): d 1.36 (t, J = 6.2 Hz, 3H), 1.58 (d,
J = 7.0 Hz, 3H), 3.62–4.46 (m, 16H), 6.00 (d, J = 7.4 Hz, 1H), 7.72 (d, J = 7.4 Hz,
0.3H), 7.77 (d, J = 7.4 Hz, 0.7H). 31P NMR (202 MHz, CD3OD): d 22.61 (s, 0.74P),
23.09 (s, 0.26P). HRMS-FAB m/z [M+H]+ calcd for C17H30N5O9P: 480.1859.
Acknowledgments
Found: 480.1863. Anal. calcd for
C
17H30N5O9PÁ3(C2HO2F3)Á0.5(C4H10O) C,
This work was supported by grants AI061457 and AI091216
from the National Institutes of Health. L.W.P. was a 2007–2008
Stauffer Fellow and a 2008–2009 WiSE Merit Fellow.
34.97%; H, 4.46%; N, 8.16%. Found: C, 34.68%; H, 4.22%; N, 8.09%. Compound
5: 1H NMR (400 MHz, CD3OD): d 1.12 and 1.14 (d, J = 6.7 Hz, 6H), 1.33–1.37 (m,
3H), 2.29 (m, 1H), 3.60–4.46 (m, 16H), 5.93 (d, J = 7.3 Hz, 1H), 7.61 (d, J = 7.3 Hz,
0.3H), 7.65 (d, J = 7.3 Hz, 0.7H). 31P NMR (162 MHz, CD3OD): d 22.60 (s, 0.65P),
23.17 (s, 0.35P). HRMS–FAB m/z [M+H]+ calcd for C19H34N5O9P: 508.2172.
Found: 508.2180.
Supplementary data
28. Wan, W. B.; Beadle, J. R.; Hartline, C.; Kern, E. R.; Ciesla, S. L.; Valiaeva, N.;
Hostetler, K. Y. Antimicrob. Agents Chemother. 2005, 49, 656.
29. Compound 6: 1H NMR (400 MHz, D2O): d 0.95 (d, J = 6.9 Hz, 6H), 1.11 and 1.13
(2d, J = 6.2 Hz, 6H), 2.12 (m, 1H), 3.43–3.49 (m, 2H), 3.59–3.77 (m, 5H), 3.90–
4.01 (m, 2H), 4.05–4.11 (m, 1H), 4.57 (m, 1H), 4.91 (sept, 1H), 5.90 (d, J = 6.9 Hz,
1H), 7.53 (d, J = 7.4 Hz, 1H). 31P NMR (162 MHz, CD3OD): d 17.32 (s, 1P). HRMS-
FAB m/z [M+H]+calcd for C19H34N5O9P: 508.2167. Found: 508.2168. LC–MS:
Rt = 14.92 min; [M+H]+ 508.1. Compound 7: 1H NMR (400 MHz, D2O): d 0.91
and 0.92 (2d, J = 6.8 Hz, 6H), 1.12 and 1.14 (2d, J = 6.2 Hz, 6H), 2.05–2.17 (m,
1H), 3.46–3.52 (m, 2H), 3.60–3.79 (m, 5H), 3.99–4.05 (m, 2H), 4.10–4.16 (m,
1H), 4.51–4.53 (m, 1H), 4.93 (sept, 1H), 6.04 (d, J = 7.8 Hz, 1H), 7.73 (d,
J = 7.8 Hz, 1H). 31P NMR (162 MHz, D2O): d 16.90. HRMS–FAB m/z [M+H]+ calcd
for C19H34N5O9P: 508.2167. Found: 508.2172.
Supplementary data (experimental details, including synthesis
of prodrugs, oral bioavailability and antiviral assays, and 1H and
31P NMR spectra for intermediates and compounds 4, 5, 6 and 7)
associated with this article can be found in the online version at
doi:10.1016/j.bmcl.2011.04.126.
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