Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Article abstract of DOI:10.1016/j.bmcl.2006.11.012

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with

Full text of DOI:10.1016/j.bmcl.2006.11.012

Bioorganic & Medicinal Chemistry Letters 17 (2007) 583–586
Synthesis and biological activation of an ethylene glycol-linked
amino acid conjugate of cyclic cidofovir
Ulrika Eriksson,a,b John M. Hilfinger,c Jae-Seung Kim,c Stefanie Mitchell,c
Paul Kijek,c Katherine Z. Borysko,b Julie M. Breitenbach,b John C. Drach,b
Boris A. Kashemirova and Charles E. McKennaa,*
aDepartment of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA
bCollege of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
cTSRL, Inc. Ann Arbor, MI 48108, USA
Received 15 October 2006; revised 3 November 2006; accepted 6 November 2006
Available online 10 November 2006
Abstract—Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited
by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe)
with the cyclic form of cidofovir (cHPMPC) via a P–O ester bond were synthesized and their pH-dependent stability (3 and 4),
potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between
pH 3 and 5, but underwent rapid hydrolysis in liver (t1/2 = 3.7 min), intestinal (t1/2 = 12.5 min), and Caco-2 cell homogenates
(t1/2 = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4· more active than ganciclovir
(IC50 value, 0.68 lM vs 3.0 lM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar
to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly
discussed.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Cidofovir (Vistideꢁ, HPMPC, 1, Fig. 1) is a broad-spec-
NH2
N
NH2
N
trum anti-viral agent that is used in the treatment of
cytomegalovirus (CMV) retinitis in AIDS patients.
Cidofovir also has therapeutic potential in the treatment
of other herpes and DNA viruses including polyoma-,
papilloma-, adeno-, and poxvirus infections.1,2 Recently,
cidofovir has become of particular interest as a potential
emergency therapy for orthopox virus infections.3 An
important limitation of cidofovir and analogous nucleo-
tide drugs in a therapeutic role is their low oral bioavail-
ability and poor transport into cells, necessitating
intravenous administration. In an emergency situation
such as a smallpox outbreak, an orally available form
of cidofovir would thus be highly advantageous.
N
N
O
O
O
OH
OH
O
O
P
O
OH
P
O
OH
2
1
Figure 1. Chemical structures of cidofovir (HPMPC) 1 and cyclic
cidofovir (cHPMPC) 2.
which substantially accounts for its low bioavailability
(<5%).4,5 Cyclic cidofovir (cHPMPC, 2, Fig. 1) under-
goes biotransformation when exposed to cCMP phos-
phodiesterase to generate the parent drug, HPMPC.6,7
Severe nephrotoxic effects have been associated with
cidofovir treatment8, while cHPMPC has been reported
to be significantly less nephrotoxic.6 cHPMPC also
exhibits low oral bioavailability, indicating a need to
mask its remaining anionic charge at physiological pH.
Our current research is focused on improving cidofovir’s
oral bioavailability. The phosphonic diacid group of
cidofovir is ionized under physiological conditions,
Keywords: Prodrug; cHPMPC; HPMPC; Biotransformation; HCMV;
Anti-viral; Biodefense.
Prodrugs9 have proven valuable for increasing drug bio-
availability as well as for drug targeting after internal
absorption.10 The prodrug strategy is based on chemical
*
Corresponding author. Tel.: +1 213 740 7007; fax: +1 213 740
0930; e-mail: mckenna@ usc.edu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.012

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U. Eriksson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 583–586
modification of the drug, often achieved by conjugating
a pro-moiety to the pharmacophore. The modification
should not only confer enhanced transport, but must
also be reversible via biotransformation in vivo to
release the parent drug.
Scheme 1. Synthesis of ethylene glycol-linked amino acids 6 and 7.
Reagents and conditions: (i) DMAP (0.15 equiv), DCC (1.3 equiv),
CH2Cl2, rt, 18 h, 48–84%.
We have previously reported the synthesis of dipeptide
conjugates of cidofovir as prodrugs targeting a peptide
transporter (PepT1) present in the small intestine.11–13
Several of these conjugates showed significantly
enhanced intestinal uptake in a rat model.11–13 Gly-
sar, a known PepT1 substrate, inhibited uptake in the
same model. Attaching the pro-moiety of the prodrug
through the phosphonic acid group on cyclic cidofovir
achieves complete masking of cidofovir’s original
phosphonic diacid group. On exposure of these cyclic
cidofovir dipeptide conjugates to biological fluids, the
parent drug was rapidly released from the biologically
benign dipeptide pro-moiety.11–13 Other research groups
have recently published different types of cidofovir
prodrugs designed for enhanced absorption in the
intestine,14,15 such as ether-lipid-conjugates of cidofovir
and cyclic cidofovir, in which the pro-moiety resembles
a natural lipid.16 Alternative prodrug approaches have
been documented in the literature.17–21
After purification of 6 (48%) and 7 (84%), by silica gel
column chromatography, the ethylene glycol-linked
amino acids were conjugated to cidofovir using 2.5
equiv PyBOP as the condensing agent (Scheme 2). Con-
current conversion of HPMPC to cHPMPC occurs in a
1-pot reaction.11 The reaction is easily monitored by
31P NMR: cyclic cidofovir has a chemical shift of
ꢀ5 ppm, while the ethylene glycol-linked amino acid
conjugates show resonance between 13 and 15 ppm.
The t-Boc-protected conjugates (8 and 9) were purified
by silica gel column chromatography (CH2Cl2/acetone/
methanol, 6:3:1) and deprotected by exposure to triflu-
oroacetic acid (TFA). The final compounds, 3 and
4, were obtained as
a diastereoisomeric mixture
(diastereomeric ratios of 1:0.8), after purification by
preparative TLC on silica in overall yields of 8% and
25%, respectively.26,27
The successful valyl ester prodrug of acyclovir (valacy-
clovir)22 stimulated our interest in exploring the chemi-
cal and biological properties of a single amino acid
conjugate of cyclic cidofovir. Several structural require-
ments must be satisfied in masking the phosphonic
diacid group of cidofovir and targeting the active trans-
porter PepT1 present in the small intestine. One crucial
structural requirement for optimal recognition by PepT1
is a free –NH2 group.23,24 Therefore, the synthetic strat-
egy presented herein involves P–O conjugation between
cyclic cidofovir and an ethylene glycol ester-linked ami-
no acid, which masks the negative charge on the cyclic
cidofovir (and the amino acid carboxyl group), leaving
the N-terminal of the amino acid free for possible
involvement as a substrate recognition site for PepT1
(Fig. 2).
The stability of cHPMPC, 3, and 4 in buffered aqueous
solution at 37 ꢂC was evaluated by incubating each
compound (500 lM) in 50 mM phosphate buffer in
which the pH was adjusted by HCl to 3.0, 4.0, 5.0,
6.0, or 7.0. Aliquots (0.25 mL) were removed at 0, 1,
2, and 3 h and immediately combined with an equal
volume of ice-cold 10% TFA to minimize degradation
prior to HPLC analysis. The rate of disappearance
was estimated by HPLC and the half-life (t1/2) was
determined from apparent first order rate constant
derived from linear regression analysis (r2 6 0.95) of
pseudo-first order plots of prodrug concentration
versus time (Table 1).
The ethylene glycol-linked valine and phenylalanine
conjugates of cyclic cidofovir (3 and 4) showed no evi-
dence of hydrolysis over 3 h at pH values 65.0.
Although 3 and 4 were stable in moderately acidic solu-
tions (3.0–5.0), noticeable but slow hydrolysis was ob-
served at pH 6.0–7.0. These observations were in
agreement with the chemical stability observed for the
cyclic cidofovir dipeptide derivatives previously report-
ed.11 Further investigations focused on the L-Val conju-
gate 3. The activation of 3 in the presence of endogenous
hydrolases was investigated by exposing the compound
(control cHPMPC) to cellular (Caco-2 and HFF) and
tissue homogenates (rat liver and intestine) as well as
to an intestinal perfusate obtained from Sprague–Daw-
ley rats. cHPMPC and 3 were exposed to the various
homogenates at 37 ꢂC and samples taken at several time
intervals between 0 and 3 h were analyzed by HPLC.
The half-lives of cHPMPC and 3 when exposed to
homogenates were determined as described for the
buffer pH stability data. The results are summarized in
Table 2.
(S)-HPMPC was synthesized by modification11 of a pre-
viously described25 method. The ethylene glycol-linked
amino acids were synthesized by conjugating t-Boc-pro-
tected amino acids (L-Val, 3, or L-Phe, 4, 1 equiv) to
ethylene glycol (4 equiv) using DCC in the presence of
a catalytic amount of DMAP (Scheme 1).
NH2
N
O
N
O
O
O
O
P
O
O
NH2
R
3 R=CH(CH3)2
4 R=CH2Ph
Figure 2. Structure of the synthesized ethylene glycol-linked amino
acid conjugates of cyclic cidofovir 3 and 4.

U. Eriksson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 583–586
585
Scheme 2. Synthesis of 3 and 4: ethylene glycol-linked amino acid conjugates of cyclic cidofovir. Reagents and conditions: (i) PyBOP (2.5 equiv),
DIEA, DMF, 40 ꢂC, 2.5–4 h, 30–56%; (ii) TFA, CH2Cl2, rt 4 h.
to duplicate wells at 4–8 concentrations. After incuba-
tion at 37 ꢂC for 7–10 days, cell sheets were fixed,
stained with crystal violet, and the microscopic plaques
enumerated.29 Drug effects were calculated as a percent-
age of reduction in the number of plaques present at
each drug concentration compared to the number ob-
served in the absence of the drug. Fifty percent inhibito-
ry concentrations (IC50) were calculated from the linear
portions of the regression lines and are presented in
Table 3.30 Compared to the control drug (ganciclovir),
3 was found to be fourfold more potent.
Table 1. Half-lives (t1/2 in hours) for cHPMPC, 3 and 4 presented in
hours at various pHsa
t1/2(h)
pH 3.0
pH 4.0
pH 5.0
pH 6.0
pH 7.0
cHPMPC
st
st
st
st
st
st
st
st
st
st
st
3
4
16.0
nd
3.5
3.7b
a t1/2 of 500 lM cHPMPC, 3 and 4 in an aqueous solution at 37 ꢂC as a
function of pH. (st, stable; nd, not done.)
b pH 6.5.
The cytotoxicity of 3 was evaluated in KB cells. Cells
were grown logarithmically for two days in the presence
of each compound (in triplicate) and evaluated by spec-
trophotometric quantitation of crystal violet dye eluted
from stained KB cells.29 It was found that cHPMPC
as well as 3 showed little to no cytotoxicity in KB cells
up to a concentration of 100 lM.
Table 2. Half-lives (t1/2 in minutes) for HPMPC and 3 in cellular and
tissue homogenatesa
a
t1/2 (min)
Caco-2
HFF
Liver
Intestine
Perfusateb
cHPMPC
3
st
20.2
st
69.9
st
3.7
st
12.5
st
141.4
a Estimated t1/2 of cidofovir and 3 in homogenate solutions at 37 ꢂC.
(st, stable.)
b Perfusate represents intestinal luminal contents and is prepared by
passing buffered solution (pH 6.5) through an isolated intestinal
The HPMPC compounds were next tested directly for
oral availability by direct injection of the drugs into
the gastrointestinal tract of rat (Table 4). Compound 3
is efficiently converted to cHPMPC in this in vivo
system: >90% of the sample detected after dosing of 3
was cHPMPC. After direct injection of 3 into the GI
tract of rat, the oral availability of 3 was estimated to
be 1.8%, which was not significantly different from
cHPMPC (2.2%). Since improved oral bioavailability
was not achieved, separation and evaluation of the 3
individual diastereoisomers were not pursued.
When the t1/2 values for 3 obtained from the buffer pH
and homogenate stability studies are compared, it is
apparent that although 3 is stable in moderately acidic
conditions and only slowly hydrolyzed in neutral buffer,
it is rapidly metabolized by endogenous liver, intestinal,
and cellular enzymes. The activation pathway for 3 and
the relevant enzymes involved are not yet known.
In conclusion, we have synthesized two novel ethylene
glycol-linked amino acid conjugates of cyclic cidofovir.
To obtain a marker for its anti-viral activity, 3 was eval-
uated in a HCMV plaque reduction assay using the
Towne strain, plaque purified isolate Po of HCMV
(kindly provided by Dr. Mark Stinski, University of
Iowa). HFF cells seeded in 24-well cluster dishes were
infected with approximately 100 pfu of HCMV per
cm2 cell sheet. Following virus adsorption, the com-
pounds, prepared as 10 mg/mL stock solutions in
DMSO, were diluted with growth medium and added
Table 3. IC50 data for HPMPC and 3 obtained in a HCMV plaque
reduction assay conducted in HFF cells
Drug
IC50 (lM)
3
0.68
3.0
Controla
a Ganciclovir served as the control.

586
U. Eriksson et al. / Bioorg. Med. Chem. Lett. 17 (2007) 583–586
Table 4. Bioavailability of HPMPC and 3 conducted in rat
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Dose normalized
mean AUC0–1 (ng/mL h)
% Bioavailabilitya
cHPMPC-iv
3
cHPMPC
472.2
258.5
304.6
100
1.8
2.2
a The AUC for 3 was determined by combining the contributions from
both the generated cHPMPC and the prodrug. The bioavailability
was calculated from the ratio of the dose normalized oral AUC
divided by the intravenously (iv) administered cHPMPC AUC data,
and a factor to account for the difference in the iv dose versus oral
dose (0.1 mg iv vs 3 mg oral).
One, the valine conjugate (3) was investigated in detail.
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ent drug in vivo. Compound 3 was also highly active
against HCMV in a HFF cell assay showing an IC50 value
in the submicromolar range, fourfold lower than ganci-
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toxic at a concentration of 100 lM when evaluated in
KB cells. However, the estimated oral bioavailability in
rat of 3 was similar to that of the parent drug. This indi-
cated that this ethylene glycol-linked mono-L-Val deriva-
tive of cHPMPC, while susceptible to biotransformation
in vivo, lacks a structural requirement for enhanced oral
bioavailability that is present in the Val-Ser-side chain-
linked dipeptide conjugates of cHPMPC.11
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Acknowledgments
We thank the NIH for financial support (R43-
AI056864). This research was presented in part at the
19th International Conference on Antiviral Research
(ICAR), San Juan, Puerto Rico, May 7th–11th 2006.31
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Supplementary data
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Vemishetti, P. Tetrahedron Lett. 1994, 35, 3243.
26. Compound 3: 1H NMR (CD3OD, ppm): 0.98 (6H, m),
2.21 (1H, m), 3.67–4.43 (12H, m), 5.85 (1H, m), 7.54–7.62
(1H, 2d, J = 7.1). 31P NMR (CD3OD, ppm): 13.91 (1P, s)
and 15.16 (1P0, s). Compound 4: 1H NMR (CD3OD,
ppm): 3.04–4.40 (14H, m), 5.82 (1H, d, J = 7.2), 5.86 (1H,
d, J = 8.28), 7.15–7.30 (5H, m), 7.53 (1H, d, J = 7.2), 7.63
(1H, d, J = 8.28). 31P NMR (CD3OD, ppm): 13.87 (1P, s)
and 15.23 (1P0, s).
(I) Detailed synthetic procedures for compounds 3 and
4, (II) NMR spectra for 3 and 4, and (III) methodology
for evaluation of the oral bioavailability of 3. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2006.11.012.
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