Alkoxyalkyl esters of cidofovir and cyclic cidofovir exhibit multiple-log enhancement of antiviral activity against cytomegalovirus and herpesvirus replication in vitro

Article abstract of DOI:10.1128/AAC.46.8.2381-2386.2002

The incidence of cytomegalovirus (CMV) retinitis is declining in AIDS patients but remains a significant clinical problem in patients with organ transplants and bone marrow transplants. Prophylaxis with ganciclovir (GCV) or valganciclovir reduces the incidence of CMV disease but may lead to the emergence of drug-resistant virus with mutations in the UL97 or UL54 gene. It would be useful to have other types of oral therapy for CMV disease. We synthesized hexadecyloxypropyl and octadecyloxyethyl derivatives of cyclic cidofovir (cCDV) and cidofovir (CDV) and found that these novel analogs had 2.5- to 4-log increases in antiviral activity against CMV compared to the activities of unmodified CDV and cCDV. Multiple-log increases in activity were noted against laboratory CMV strains and various CMV clinical isolates including GCV-resistant strains with mutations in the UL97 and UL54 genes. Preliminary cell studies suggest that the increase in antiviral activity may be partially explained by a much greater cell penetration of the novel analogs. 1-O-Hexadecyloxypropyl-CDV, 1-O-octadecyloxyethyl-CDV, and their corresponding cCDV analogs are worthy of further preclinical evaluation for treatment and prevention of CMV and herpes simplex virus infections in humans.

Full text of DOI:10.1128/AAC.46.8.2381-2386.2002

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2002, p. 2381–2386
0066-4804/02/$04.00ϩ0 DOI: 10.1128/AAC.46.8.2381–2386.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 8
Alkoxyalkyl Esters of Cidofovir and Cyclic Cidofovir Exhibit
Multiple-Log Enhancement of Antiviral Activity against
Cytomegalovirus and Herpesvirus Replication In Vitro
James R. Beadle,1 Caroll Hartline,2 Kathy A. Aldern,1 Natalie Rodriguez,1
Emma Harden,2 Earl R. Kern,2 and Karl Y. Hostetler1*
Department of Medicine, Veterans Affairs Medical Center, and the University of California, San Diego,
La Jolla, California 92093-0676,1 and Department of Pediatrics, University of Alabama
at Birmingham, Birmingham, Alabama 35294-21702
Received 16 November 2001/Returned for modification 5 February 2002/Accepted 18 April 2002
The incidence of cytomegalovirus (CMV) retinitis is declining in AIDS patients but remains a significant
clinical problem in patients with organ transplants and bone marrow transplants. Prophylaxis with ganciclovir
(GCV) or valganciclovir reduces the incidence of CMV disease but may lead to the emergence of drug-resistant
virus with mutations in the UL97 or UL54 gene. It would be useful to have other types of oral therapy for CMV
disease. We synthesized hexadecyloxypropyl and octadecyloxyethyl derivatives of cyclic cidofovir (cCDV) and
cidofovir (CDV) and found that these novel analogs had 2.5- to 4-log increases in antiviral activity against CMV
compared to the activities of unmodified CDV and cCDV. Multiple-log increases in activity were noted against
laboratory CMV strains and various CMV clinical isolates including GCV-resistant strains with mutations in
the UL97 and UL54 genes. Preliminary cell studies suggest that the increase in antiviral activity may be
partially explained by a much greater cell penetration of the novel analogs. 1-O-Hexadecyloxypropyl-CDV,
1-O-octadecyloxyethyl-CDV, and their corresponding cCDV analogs are worthy of further preclinical evalua-
tion for treatment and prevention of CMV and herpes simplex virus infections in humans.
Although the incidence and prevalence of cytomegalovirus
(CMV) retinitis in AIDS patients are declining due to the use
of highly active antiretroviral therapies (12), CMV continues
to be a major cause of opportunistic infections in patients with
allogeneic bone marrow transplants (BMTs) and solid-organ
transplants (6). In transplant patients, the incidence of CMV
infection increases with the duration and degree of immuno-
suppression, approximating 70% in allogeneic BMT patients
who are CMV seropositive (2) and in patients receiving solid-
organ transplants from CMV-seropositive donors (4, 18).
CMV disease is associated with a high risk of morbidity and
mortality in solid-organ transplant and allogeneic BMT pa-
tients (6). While prophylaxis with ganciclovir (GCV) signifi-
cantly reduces the incidence of CMV disease in transplant
recipients, drug resistance may emerge because of mutations in
the UL97 gene, which catalyzes the initial phosphorylation of
GCV, or in the UL54 polymerase gene of the virus (for a
review, see reference 5). Current therapies for CMV disease in
transplant patients are based primarily on intravenous therapy
with GCV, cidofovir (CDV), or foscarnet (phosphonoformate)
or, more recently, with oral valganciclovir.
glyceryl residues to the phosphate of ACV monophosphate or
GCV monophosphate (1, 8, 9). These ether lipid analogs gen-
erally show severalfold increases in activity over the activity of
underivatized ACV or GCV and provide increased oral ab-
sorption in rodents (8). In woodchucks with hepatitis, 1-O-
hexadecyloxypropyl-phospho-ACV reduced woodchuck hepa-
titis virus DNA levels in plasma by nearly 2 logs after 4 weeks
of treatment with 10 mg/kg of body weight twice daily, but a
five times greater oral dose of ACV (molar basis) had no effect
(7).
To determine if more effective and less toxic forms of CDV
or cyclic CDV (cCDV) can be designed, we synthesized several
alkoxyalkyl analogs of these compounds and evaluated their
antiviral activities against human CMV (HCMV) and herpes
simplex virus (HSV) by DNA reduction and plaque reduction
assays with cells infected with various wild-type and GCV-
resistant strains of CMV and HSV type 1 (HSV-1). Surpris-
ingly, we detected multiple-log enhancement of the in vitro
antiviral activities of the alkoxyalkyl analogs compared with the
activity of underivatized cCDV or CDV.
It would be useful to identify more effective oral therapies
for the treatment of CMV disease in allogeneic bone marrow,
stem cell, or solid-organ transplant patients and in CMV ret-
initis patients with AIDS. We have developed a strategy to
improve the antiviral activity and oral absorption of acyclovir
(ACV) and GCV by covalently attaching alkoxyalkyl or alkoxy-
MATERIALS AND METHODS
Chemistry. (i) General. All products were homogeneous by thin-layer chro-
matography (TLC), performed on Analtech 250-␮m Silica Gel GF Uniplates and
visualized under UV light with phospray (Supelco, Bellafonte, Pa.) and by char-
ring. Chromatographic purification was done by the flash method with Merck
silica gel 60 (240 to 400 mesh). 1H and 31P nuclear magnetic resonance (NMR)
spectra were recorded at 400 MHz on a Varian HG-400 spectrophotometer with
tetramethylsilane (internal) and 85% D3PO4 in D2O (external) as references for
1H and 31P (0.00 ppm), respectively. Electrospray ionization mass spectroscopy
(ESI) was performed by Mass Consortium (San Diego, Calif.). CDV (compound
1) was provided by Gilead Sciences, Inc. (Foster City, Calif.). The synthesis and
* Corresponding author. Mailing address: Department of Medicine
(0676), University of California, 305 Clinical Sciences Building, 9500
Gilman Dr., San Diego, La Jolla, CA 92093-0676. Phone: (858) 552-
8585, ext. 2616. Fax: (858) 534-6133. E-mail: khostetl@ucsd.edu.
2381

2382
BEADLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Synthesis of alkoxyalkyl analogs of CDV and cCDV. Reagents: a, N,N-dicyclohexyl-morpholinocarboxamidine, 1,3-dicyclohexylcarbo-
diimide, pyridine, 100°C; b, ODE-Br, ODP-Br, or HDP-Br, N,N-dimethylformamide, 80°C; c, 0.5 M NaOH. The underscored numbers in
parentheses correspond to the compound numbers used throughout the report.
characterization of compounds 2, 4, 5, 7, and 8 have been reported previously
(10) (Fig. 1 ).
tography. The product (133 mg, 58%) was eluted with CH2Cl2-MeOH (70:30).
1H NMR ␦ (DMSO-d6) 0.86 (t, 3-H), 1.24 (broad s, 30-H), 1.47 (m, 2-H), 1.73 (p,
2-H), 3.20 to 3.89 (m, 13-H), 5.72 (m, 1-H), 7.21 (d, 2-H), 7.54 (d, 1-H); 31P NMR
␦ ϩ13.98; MS (ESI) m/z 584 (Mϩ Na)ϩ, 560 (MϪ H)Ϫ. TLC Rf ϭ 0.27 (CHCl3-
MeOH-concentrated NH4OH-H2O; 80:20:1:1).
1-Bromo-3-octadecyloxypropane. Triphenylphosphine (10.0 g, 38 mmol) was
added to a cooled (0°C) solution of 3-octadecyloxy-1-propanol (5.0 g, 15 mmol)
and carbon tetrabromide (10.6 g, 32 mmol) in dichloromethane (70 ml) in 2-g
portions over 30 min. The reaction mixture was stirred for 45 min at 0°C and then
for 1 h at room temperature. The reaction mixture was concentrated, and the
residue was dissolved in ether. After the mixture was stirred for 1 h, it was filtered
and the filtrate was concentrated. The residue was purified by flash chromatog-
raphy. Elution with 90% hexane–10% ethyl acetate yielded 4.3 g (77%) as a
colorless oil. 1H NMR ␦ 0.88 (t, 3-H), 1.25 (br s, 30-H), 1.56 (m, 2-H), 2.09 (p,
2-H), 3.42 (t, 2-H), 3.49 to 3.54 (two triplets, 4-H). Electrospray mass spectros-
copy (MS), positive and negative, failed to give a molecular ion.
Preparation of control and drug-containing liposomes for antiviral assays.
For the in vitro studies, 1-O-hexadecyloxypropyl-cCDV (HDP-cCDV), 1-O-hexa-
decyloxypropyl-CDV (HDP-CDV), 1-O-octadecyloxyethyl-cCDV (ODE-cCDV),
1-O-octadecyloxyethyl-CDV (ODE-CDV), 1-O-octadecyloxypropyl-cCDV (ODP-
cCDV), 1-O-octadecyloxypropyl-CDV (ODP-CDV), and hexadecyl-cCDV
(HD-cCDV) were incorporated into liposomes. Chloroform solutions of the
phospholipids, cholesterol (CH), and drugs were mixed to provide dioleoylphos-
phatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), CH, and an
alkoxyalkanol-CDV or alkoxyalkanol-cCDV analog at a molar ratio of 50/10/30/
10. Control liposomes were prepared without drug and had a DOPC-DOPG-CH
composition of 60/10/30. The chloroform was removed under a stream of nitro-
gen, and the thin lipid film was hydrated by the addition of 360 ␮l of 250 mM
sorbitol–20 mM sodium acetate (pH 5.5). The small multiple-dose vial was sealed
under nitrogen with a Teflon-lined cap and sonicated for 1 h at 42°C. The clear
preparation of sonicated liposomes, representing a nominal drug concentration
of 5 mM, was diluted sequentially with Dulbecco’s modified Eagle’s medium
containing 4% fetal bovine serum to provide the indicated range of concentra-
tions, and the medium was added to the virus-infected cells as indicated below.
Antiviral assays for activities against various strains of CMV and HSV-1 in
vitro. Antiviral activity against HCMV (AD169) or HSV-1 was determined by a
DNA reduction assay with MRC-5 human lung fibroblast cells with DNA probes
supplied by Diagnostic Hybrids, Athens, Ohio, as described previously (9) or by
plaque reduction assay with human foreskin fibroblast cells infected with various
strains of HCMV or HSV-1 (11). The results of antiviral assays with HDP-CDV
presented to cells in dilute DMSO were similar to those obtained with the
compound presented to cells in liposomes, and blank liposome controls had no
effect on viral replication. The antiviral activities of the various alkoxyalkyl esters
of CDV and cCDV were also determined in CMV-infected murine, rat, and
guinea pig embryonic fibroblast cells by plaque reduction assays (11, 14). The
cytotoxic concentration of drug which reduced the viable cell number by 50%
(CC50) was determined. In the plaque reduction assays, cytotoxicity was deter-
mined by measurement of neutral red uptake (14).
(ii) cCDV-hexadecyl ester (compound 3). A mixture of compound 2 (188 mg,
0.34 mmol) and 1-bromohexadecane (520 mg, 1.8 mmol) in N,N-dimethylform-
amide (25 ml) was stirred and heated to 80°C for 6 h. The mixture was concen-
trated, and the residue was purified by flash chromatography. Elution with 10%
methanol (MeOH)–90% CH2Cl2 yielded 58 mg of compound 3 (33%) as a white
powder. 1H NMR ␦ (dimethyl sulfoxide [DMSO]-d6) 0.85 (t, 3-H), 1.23 (broad s,
26-H), 1.60 (m, 2-H), 3.55 to 4.20 (m, 9-H), 5.6 (dd, 1-H), 7.18 and 7.04 (broad
d, 2-H), 7.57 and 7.45 (d, 1-H); 31P NMR ␦ ϩ13.60 and ϩ12.48 (mixture of axial
and equatorial diastereomers) (13); MS (ESI) m/z 486 (Mϩ H)ϩ, 484 (MϪ H)Ϫ
TLC Rf ϭ 0.9 (CHCl3-MeOH-concentrated NH4OH-H2O; 80:20:1:1).
.
(iii) cCDV-octadecyloxypropyl ester (compound 6). A mixture of compound 2
(1.02 g, 1.8 mmol) and 1-bromo-3-octadecyloxypropane (2.82 g, 7.5 mmol) in
N,N-dimethylformamide (35 ml) was stirred and heated (80°C) for 6 h. The
mixture was then concentrated in vacuo, and the residue was purified by flash
chromatography. Elution with 10% MeOH–90% CH2Cl2 afforded 450 mg of a
white powder (41% yield). High-pressure liquid chromatography, TLC, and
spectroscopic analysis showed the presence of two diastereomeric (axial and
equatorial) alkylation products. 1H NMR ␦ (DMSO-d6) 0.85 (t, 3-H), 1.23 (broad
s, 30-H), 1.47 (m, 2-H), 1.84 (p, 2-H), 3.55 to 4.20 (m,13-H), 5.70 (dd, 1-H), 7.18
and 7.04 (broad d, 2-H), 7.55 and 7.45 (d, 1-H); 31P NMR ␦ ϩ13.61 and ϩ12.31;
MS (ESI) m/z 572 (Mϩ H)ϩ, 570 (MϪ H)Ϫ. TLC Rf ϭ 0.9 (CHCl3-MeOH-
concentrated NH4OH-H2O; 80:20:1:1).
(iv) CDV-octadecyloxypropyl ester (compound 9). Compound 6 (230 mg, 0.38
mmol) was dissolved in 0.5 M NaOH (5 ml), and the mixture was stirred at room
temperature for 1.5 h. The solution was neutralized with acetic acid, and the
precipitate was isolated by filtration and then purified by flash column chroma-

VOL. 46, 2002
Compound
DRUG ACTIVITY AGAINST CMV AND HERPESVIRUS REPLICATION
2383
TABLE 1. Antiviral activities and selectivities of CDV compounds in HCMV or HSV-1-infected MRC-5 human
lung fibroblast measured by DNA reduction assaya
EC50 (␮M)
Selectivity for:
Toxicity
(CC50 [␮M])
HCMV
HSV-1
HCMV
HSV-1
CDV
0.46 Ϯ 0.08 (4)
3.3 Ϯ 3.7 (3)
0.001 Ϯ 0.002 (3)
0.0001 Ϯ 0.0001 (4)
0.003 Ϯ 0.001 (3)
Ͼ1,000
210
10
Ͼ303
2 ϫ 105
1 ϫ 105
1 ϫ 104
Ͼ2,174
10 ϫ 106
5 ϫ 106
1 ϫ 106
ODE-CDV
HDP-CDV
ODP-CDV
2 ϫ 10Ϫ5 Ϯ 3 ϫ 10Ϫ5 (6)
2 ϫ 10Ϫ6 Ϯ 3 ϫ 10Ϫ6 (4)
3 ϫ 10Ϫ5 Ϯ 4 ϫ 10Ϫ5 (4)
32
cCDV
0.47 Ϯ 0.13 (3)
0.04 Ϯ 0.01 (3)
2.3 Ϯ 1.5 (3)
3.1 Ϯ 2.4 (3)
0.005 Ϯ 0.005 (3)
0.04 Ϯ 0.03 (3)
0.35 Ϯ 0.18 (3)
Ͼ1,000
6.5
Ͼ2,128
163
Ͼ435
2.0
HD-cCDV
ODE-cCDV
HDP-cCDV
ODP-cCDV
1 ϫ 10Ϫ4 Ϯ 1 ϫ 10Ϫ4 (4)
3 ϫ 10Ϫ4 Ϯ 3 ϫ 10Ϫ4 (6)
3 ϫ 10Ϫ4 Ϯ 3 ϫ 10Ϫ4 (3)
320
3 ϫ 106
1 ϫ 106
1 ϫ 106
6 ϫ 104
8 ϫ 103
900
320
320
a The values are means Ϯ standard deviations. Numbers in parentheses represent number of replicates. Selectivity is CC50/EC50
.
RESULTS
The antiviral activities of the HDP and ODE analogs of
cCDV and CDV were also examined by the plaque reduction
assay with human foreskin fibroblast cells infected with various
laboratory strains and clinical isolates of HCMV, and the an-
tiviral activities of these compounds were compared with those
of GCV, cCDV, and CDV (Table 3). In general, when the
antiviral activities of CDV and cCDV were compared to those
of the respective HDP and ODE esters, multiple-log increases
in antiviral activities were observed. For example, for strain
AD169, the EC50 of CDV was 0.38 ␮M, whereas the EC50s of
both HDP-CDV and ODE-CDV were 0.0009 ␮M, represent-
ing increases in activity of 2.6 logs for the new analogs. Similar
results were obtained with the Towne, Davis, and C9208/5-4-2
strains of wild-type HCMV (Table 3). Although the Toledo
strain was much less sensitive to CDV (EC50, 13.8 ␮M), the
HDP-CDV and ODE-CDV analogs were both substantially
more active, with EC50s of 0.025 ␮M, representing an increase
in antiviral activity of 2.74 logs. Nearly 3-log increases in anti-
viral activities were noted with the alkoxyalkanol analogs of
CDV against cells infected with the Coffman, C8708/17-1-1,
and C9208/5-4-2 strains of CMV. Similar findings were ob-
tained with HDP-cCDV and ODE-cCDV, except that these
analogs were generally somewhat less active than the corre-
sponding analogs of CDV (Table 3).
MRC-5 human lung fibroblasts were infected with HCMV
(AD169) or HSV-1, and the antiviral activities of CDV and
cCDV were assessed by DNA reduction assay (Table 1).
Against HCMV the 50% effective concentrations (EC50s) for
CDV and cCDV were similar (0.46 to 0.47 ␮M). The alkoxy-
alkyl analogs ODE-CDV, ODP-CDV, and HDP-CDV were 4
to 5 logs more active against HCMV, with EC50s ranging from
2 ϫ 10Ϫ6 to 3 ϫ 10Ϫ5 ␮M. In cells infected with HSV-1, CDV
and cCDV reduced viral replication by 50% at 3.3 and 2.3 ␮M,
respectively. Again, the alkoxyalkyl analogs of CDV were most
active, with EC50s of 0.0001 to 0.003 ␮M. HDP-CDV was the
most active of these three compounds. The alkoxyalkyl analogs
of cCDV were less active than the corresponding CDV com-
pounds. We also synthesized the 16-carbon straight-chain alkyl
ester of cCDV, HD-cCDV, which lacks the oxygen group two
or three carbons from the ester functionality. Interestingly, this
compound is 133 to 400 times less active than ODE- or HDP-
cCDV, esters of octadecylethanediol and hexadecylpro-
panediol, respectively (Table 1). The cytotoxicities of the
alkoxyalkyl esters of CDV and cCDV in MRC-5 cells were
greater than those observed with CDV or cCDV, but the se-
lectivities of the HDP, ODE, and ODP derivatives of cCDV
and CDV against CMV or HSV-1 increased greatly because of
the marked increases in antiviral activity. In contrast, the com-
pound lacking the oxygen heteroatom in the alkyl chain, HD-
cCDV, exhibited greater toxicity and less antiviral activity (Ta-
ble 1).
We also evaluated the activities of the analogs of CDV and
cCDV against HSV-1 and HSV-2 by the plaque reduction
assay. CDV and cCDV appeared to be less active against
HSV-1 by the plaque reduction assay (Table 2) than by the
DNA reduction assay, with EC50s of 18.0 and 30.6 ␮M, respec-
tively, compared with EC50s of 3.3 and 2.3 ␮M, respectively, by
the DNA reduction assay (Table 1). The EC50s of the alkoxy-
alkyl analogs were also higher by the plaque reduction assay
than by the DNA reduction assay. Nevertheless, increases in
antiviral activity of 2.39 to 2.81 logs were noted with HDP-
CDV and ODE-CDV, respectively, compared with the activity
of unmodified CDV. Somewhat lesser increases in activity
were noted with the analogs of cCDV versus those of unmod-
ified cCDV when the activities were measured by the plaque
reduction assay (Table 2).
The activities of GCV, CDV, cCDV, and the alkoxyalkyl
esters of CDV and cCDV were also evaluated against a panel
of drug-resistant HCMV mutants kindly provided to E. R.
Kern by Karen Biron of GlaxoSmithKline, Research Triangle
Park, N.C., and Donald Coen, Boston, Mass. (Table 4). The
EC50s of GCV for GCV-resistant strains with mutations in the
TABLE 2. Activities of alkoxyalkyl esters of CDV and cCDV
against HSV-1 and HSV-2 by plaque reduction assay
EC50 (␮M)
Compound
HSV-1
HSV-2
Assay 1
Assay 2
Assay 1
Assay 2
CDV
22.5
0.08
0.03
28.4
0.9
7.9
0.04
0.012
10.6
0.25
0.2
13.2
0.13
0.03
11.2
0.35
0.28
7.9
HDP-CDV
ODE-CDV
cCDV
0.03
0.03
7.6
HDP-cCDV
ODP-cCDV
0.11
0.12
0.5

2384
BEADLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. Activities of GCV, CDV, cCDV, and alkoxyalkyl esters of CDV and cCDV against HCMV replication
measured by plaque reduction assay
EC50 (␮M)b
Isolatea
GCV
CDV
HDP-CDV
ODE-CDV
cCDV
HDP-cCDV
ODE-cCDV
AD169
2.75 Ϯ 1.6
4.3 Ϯ 0
0.38 Ϯ 0
0.0009 Ϯ 0.0001
0.0009 Ϯ 0
0.00095 Ϯ 0.00007
0.025 Ϯ 0.007
0.001 Ϯ 0
0.001 Ϯ 0
0.001 Ϯ 0
0.00085 Ϯ 0.0002
0.0009 Ϯ 0.0001
0.0009 Ϯ 0.0001
0.0009 Ϯ 0.0001
0.025 Ϯ 0.02
0.001 Ϯ 0
0.31 Ϯ 0.02
0.48 Ϯ .02
0.45 Ϯ 0.07
17.1 Ϯ 8
1.2 Ϯ 0.5
2.1 Ϯ 0.4
1.3 Ϯ 0.3
1.1 Ϯ 0.26
0.001 Ϯ 0
0.0018 Ϯ 0.001
0.001 Ϯ 0
Towne
0.4 Ϯ 0.11
0.66 Ϯ 0.3
13.8 Ϯ 7.3
0.87 Ϯ 0.15
1.1 Ϯ 0.5
0.001 Ϯ 0
Davis
Toledo
Coffman
C8708/17-1-1
C9208/3-3-1
C9208/5-4-2
5.1 Ϯ 0
0.001 Ϯ 0
0.055 Ϯ 0.03
0.0015 Ϯ 0.0007
0.0015 Ϯ 0.0007
0.00095 Ϯ 0.00007
0.00095 Ϯ 0.00007
0.001 Ϯ 0
19.6 Ϯ 7.2
4.7 Ϯ 0
2.6 Ϯ 0.9
4.25 Ϯ 1.2
1.6 Ϯ 0.42
0.055 Ϯ 0.03
0.002 Ϯ 0.0007
0.0025 Ϯ 0.002
0.001 Ϯ 0
0.001 Ϯ 0
0.95 Ϯ 0.6
0.41 Ϯ 0.09
0.001 Ϯ 0
0.001 Ϯ 0
0.001 Ϯ 0
a All isolates are wild type.
b The values are the means Ϯ standard deviations of two or more determinations.
UL97 gene were 3.7 to 16.4 times greater than the average
EC50 (3.61 ␮M) for the seven wild-type strains (Table 5). CDV
and cCDV retained nearly full activity against the strains with
mutations in the UL97 gene, and their alkoxyalkyl esters were
2.5 to 2.98 logs more active than the underivatized nucleotide
phosphonates. A mutant of CMV with a mutation in the DNA
polymerase gene (UL54), mutant GDGP53, exhibited 15 to 22
times greater resistance to cCDV and CDV and 15 times
greater resistance to GCV than the wild type. Interestingly, the
alkoxyalkyl esters of cCDV and CDV retained substantial ac-
tivities against this mutant with a mutation in the DNA poly-
merase gene; HDP-CDV and ODE-CDV both had EC50s of
0.02 ␮M, a 3.4-log increase in activity compared with that of
unmodified CDV. HDP-cCDV and ODE-cCDV were also ac-
tive, showing 2.4- to 2.5-log increases in activity compared with
that of cCDV against the mutant with the polymerase muta-
tion. A double mutant, mutant 759D100, which has mutations
in both the DNA polymerase (G987A in UL54) and in UL97
(deletion of 590 to 593 in UL97) was the mutant most resistant
to GCV, but it was somewhat less cross resistant than the
mutant with a mutation in the polymerase gene, mutant
GDGP53. The antiviral activities of the alkoxyalkanol analogs
of cCDV and CDV against mutant 759D100 were intermediate
between those against the mutant with the UL97 gene muta-
tion and the mutant with the polymerase gene mutation.
Against mutant 759D100, the HDP and ODE esters of cCDV
and CDV were 2.2 to 2.5 logs more active than the unmodified
phosphonates (Table 5).
of CDV and cCDV were 1 to 2 logs less than those noted by
DNA reduction assay. However, the activities of CDV and
cCDV were similar by the two assays. Alkyl ether analogs of
both ethanediol and propanediol were highly active. Although
we have not done extensive structure-activity analyses, both 16-
and 18-carbon alkyl ether chains were highly effective. By the
DNA reduction assay, the analogs of cCDV were less active
than the corresponding CDV compounds. Interestingly, when
cCDV was coupled directly to hexadecanol, a long-chain alco-
hol 16 carbons in length, a 2-log drop in antiviral activity was
noted, demonstrating the importance of the oxygen hetero-
atom. The oxygen heteroatom may make the analogs subject to
rapid enzymatic conversion to cCDV or CDV, precursors of
the active antiviral CDV diphosphate (CDV-PP). This must be
confirmed by metabolic studies comparing the conversion to
cCDV by using radiolabeled HD-cCDV and HDP-cCDV in-
cubated with cell homogenates or subcellular membrane frac-
tions. Preliminary studies indicate that HDP-CDV is metabo-
lized by an intracellular enzyme of the phospholipase C type
(unpublished observation).
The HDP and ODE derivatives of CDV exhibited 2.5- to
4-log increases in antiviral activity depending on the antiviral
assay used (Tables 1 and 2). The mechanism of the increased
activity remains to be determined. However, CDV enters cells
by pinocytosis, which may greatly restrict passage of unmodi-
fied drug into cells. Preliminary studies in our laboratory with
14C-labeled CDV and HDP-CDV indicate that the amount of
HDP-CDV that enters the cell is increased by several logs;
intracellular CDV-PP can easily be detected when 10 ␮M
HDP-CDV is used, but when 10 ␮M CDV is used, the intra-
cellular levels of CDV-PP are substantially lower (unpublished
data). Full assessment of the mechanisms of the increased
HDP-CDV and ODE-CDV were also highly active against a
panel of strains of CMV from animals, including CMV strains
from the mouse, rat, and guinea pig. The most active com-
pounds were HDP-CDV and ODE-CDV, with EC50s of 0.0009
to 0.005 ␮M, whereas the EC50 of unmodified CDV was 0.26
␮M. Similar trends were noted with the cCDV series of com-
pounds (Table 6).
TABLE 4. Drug-resistant HCMV isolates
Drug(s) to
Affected
gene
Refer-
ence
DISCUSSION
Isolate
which the isolate
is resistant
Mutation
The covalent addition of an alkoxyalkyl ester group to the
phosphonate of CDV or cCDV resulted in remarkable in-
creases in antiviral activities against CMV and HSV-1 in vitro.
The cytotoxicities of the analogs were also increased, but se-
lectivity (CC50/EC50) was increased substantially in most cases.
When the activities were measured by plaque reduction assay,
the increases in activities observed with the alkoxyalkyl analogs
C9209/1-4-4a
C8914-6a
GCV
UL97
UL97
UL97
M460V
3
GCV
L595F
3
C8805/37-1-1a
GDGP53b
759D100b
GCV
GCV, CDV
GCV
M460V
3, 15
16
UL54 (Pol) G987A
UL54/UL97 G987A/⌬590–93 17
a Provided by Karen Biron.
b Provided by Donald Coen.

VOL. 46, 2002
DRUG ACTIVITY AGAINST CMV AND HERPESVIRUS REPLICATION
2385
TABLE 5. Effects of GCV, CDV, cCDV, and alkoxyalkyl esters against drug-resistant isolates of HCMV by plaque reduction assaya
EC50 (␮M)
Isolate
GCV
CDV
HDP-CDV
ODE-CDV
cCDV
HDP-cCDV
ODE-cCDV
Wild typea
3.61 Ϯ 1.3
0.68 Ϯ 0.29
0.0009 Ϯ 0.0006
0.00096 Ϯ 0.00005 0.99 Ϯ 0.63 0.0011 Ϯ 0.0003 0.0015 Ϯ 0.0006
Drug-resistant mutants
C9209/1-4-4
C8914-6
C8805/37-1-1
GDGP53
759D100
59.3 Ϯ 0.2
13.5 Ϯ 2
47.4 Ϯ 1.1
54.6 Ϯ 23
177 Ϯ 28.2
2.3 Ϯ 0.28
0.99 Ϯ 0.01
0.84 Ϯ 0.2
15.7 Ϯ 14.1
2.0 Ϯ 0.56
0.003 Ϯ 0
0.0040 Ϯ 0.001
0.0010 Ϯ 0
1.95 Ϯ 0.9
0.005 Ϯ 0
0.005 Ϯ 0.001
0.001 Ϯ 0
0.06 Ϯ 0.05
0.015 Ϯ 0.007
0.0045 Ϯ 0.0007
0.0045 Ϯ 0.0007
0.0010 Ϯ 0
0.0025 Ϯ 0.0007
1.45 Ϯ 0.21
0.00095 Ϯ 0.00007 0.00095 Ϯ 0.00007 0.96 Ϯ 0.2
0.020 Ϯ 0.009
0.0065 Ϯ 0.0007
0.020 Ϯ 0.008
0.0055 Ϯ 0.0007
15.9 Ϯ 12.2
2.5 Ϯ 0.1
0.050 Ϯ 0.03
0.0150 Ϯ 0.004
a The values are the means Ϯ standard deviations for the seven wild-type strains for which the results are presented in Table 3 (the Toledo strain was omitted from
the analysis).
activities of HDP-CDV and ODE-CDV awaits determination
of comparative levels of intracellular CDV monophosphate
and CDV-PP in cells incubated with equimolar concentrations
of 14C-labeled CDV and the respective analogs. However, our
preliminary studies suggest that enhanced cell uptake is a ma-
jor factor in the 2.5- to 4-log increases in antiviral activity which
have been documented.
were observed with a double mutant with mutations in both the
UL97 and the UL54 genes.
HDP-CDV and ODE-CDV are also highly active against
orthopoxviruses such as vaccinia virus and cowpox virus (10)
and monkeypox virus and smallpox virus (John Huggins, per-
sonal communication). The EC50s of HDP-CDV and ODE-
CDV for the various poxviruses are in the range of 0.01 to 0.8
␮M, making these agents of interest as potential treatments for
smallpox, should the disease reappear.
In conclusion, long-chain alkyl ethers of propanediol or
ethanediol covalently linked to cCDV or CDV provide multi-
ple-log increases in antiviral activity against laboratory wild-
type strains, various clinical isolates, and GCV-resistant strains
of HCMV in vitro. The most active compounds were HDP-
CDV and ODE-CDV, with EC50s of 2 ϫ 10Ϫ6 and 2 ϫ 10Ϫ5
␮M, respectively. Based on our previous research, compounds
of this type may be orally bioavailable (6–8). Further evalua-
tion of this approach is warranted to assess the suitability of
HDP-CDV and ODE-CDV for further development for the
treatment or prevention of human infections with the herpes-
virus group of viruses and poxviruses.
The alkoxyalkyl analogs of CDV and cCDV also showed
multiple-log increases in antiviral activities against multiple
clinical isolates of HCMV (Table 3). One of these isolates,
Towne, exhibited reduced susceptibility to GCV, cCDV, and
CDV, with EC50s 3 to 10 times greater than those for labora-
tory HCMV and clinical isolates. Although the Towne strain
was also relatively resistant to the alkoxyalkyl analogs of CDV,
the EC50s were still low (0.025 to 0.055 ␮M) and were more
than 2.5 logs lower than those of underivatized CDV or cCDV.
In addition, all of the alkoxyalkyl analogs tested had multiple-
log increases in activity compared with that of CDV against
GCV-resistant HCMV mutants (Table 5). Most of these
strains have mutations in the UL97 gene, which controls the
phosphorylation of GCV. Interestingly, a DNA polymerase
mutant, mutant GDGP53, which exhibits about 10-fold greater
resistance to CDV and cCDV and 50-fold greater resistance to
GCV than the wild type, remains sensitive to HDP-CDV and
ODE-CDV, with EC50s of 0.02 ␮M (Table 5). Although this
represents a 20-fold decrease in antiviral activity compared
with that of wild-type strains of HCMV, HDP-CDV may still
be useful against mutants of this type in vivo. Similar results
ACKNOWLEDGMENTS
This work was supported in part by NIH grants EY11834, DAMD
17-01-2-0071, AI41928, and AI-29164; by the Research Center for
AIDS and HIV Infection of the San Diego Veterans Affairs Medical
Center; and by Public Health Service contracts NO1-AI-85347 and
NO1-AI-15439 from the National Institutes of Allergy and Infectious
Diseases, NIH, Bethesda, Md.
REFERENCES
1. Beadle, J. R., G. D. Kini, K. A. Aldern, M. F. Gardner, K. N. Wright, R. J.
Ryback, E. R. Kern, and K. Y. Hostetler. 2000. Synthesis and antiviral
evaluation of 1-O-hexadecylpropane-diol-3-P-acyclovir: efficacy against
HSV-1 infection in mice. Nucleosides Nucleotides 19:471–480.
2. Boeckh, M., T. A. Gooley, D. Myerson, T. Cunningham, G. Schoch, and R. A.
Bowden. 1996. Cytomegalovirus pp65 antigenemia-guided early treatment
with ganciclovir versus ganciclovir at engraftment after allogenic marrow
transplantation: a randomized double-blind study. Blood 88:4063–4071.
3. Chou, S., A. Erice, M. C. Jordan, G. M. Vercellotti, K. R. Michels, C. L.
Talarico, S. C. Stanat, and K. K. Biron. 1995. Analysis of the UL97 phos-
photransferase coding sequence in clinical cytomegalovirus isolates and
identification of mutations conferring ganciclovir resistance. J. Infect. Dis.
171:576–583.
4. Dunn, D. L., K. J. Gillingham, M. A. Kramer, W. J. Schmidt, A. Enrice, H. H.
Balfour, Jr., P. F. Gores, R. W. Gruessner, A. J. Matas, W. D. Payne, D. E. R.
Sutherland, and J. S. Najarian. 1994. A prospective randomized study of
acyclovir versus ganciclovir plus human immune globulin prophylaxis of
cytomegalovirus infection after solid organ transplant. Transplantation 57:
876–884.
TABLE 6. Activities of alkoxyalkyl esters of CDV and cCDV
against murine, rat, and guinea pig CMV strains
EC50 (␮M)a
Compound
MCMV
RCMV
GpCMV
GCV
9.0 Ϯ 4.9
48.2 Ϯ 1.1
239.0 Ϯ 10.6
CDV
HDP-CDV
ODE-CDV
0.26 Ϯ 0.02
0.0009 Ϯ 0
0.001 Ϯ 0
0.30 Ϯ 0.03
0.004 Ϯ 0.001
0.005 Ϯ 0
0.31 Ϯ 0.003
0.0009 Ϯ 0.00007
0.0009 Ϯ 0.00007
cCDV
HDP-cCDV
ODE-cCDV
0.39 Ϯ 0.05
0.003 Ϯ 0
0.004 Ϯ 0
0.46 Ϯ 0.05
0.005 Ϯ 0
0.005 Ϯ 0
0.50 Ϯ 0.26
0.001 Ϯ 0
0.001 Ϯ 0
a The values are the means Ϯ standard deviations of two assays. Abbreviations:
MCMV, murine CMV; RCMV, rat CMV; GpCMV, guinea pig CMV.
5. Erice, A. 1999. Resistance of human cytomegalovirus to antiviral drugs. Clin.
Microbiol. Rev. 12:286–297.

2386
BEADLE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
6. Hebart, H., L. Kanz, G. Jahn, and H. Einsele. 1998. Management of CMV
infection after solid organ or stem-cell transplantation: current guidelines
and future prospects. Drugs 55:59–72.
7. Hostetler, K. Y., J. R. Beadle, W. E. Hornbuckle, C. A. Bellezza, I. A.
Tochkov, P. J. Cote, J. L. Gerin, B. E. Korba, and B. C. Tennant. 2000.
Antiviral activities of oral 1-O-hexadecylpropanediol-3-phosphoacyclovir
and acyclovir in woodchucks with chronic woodchuck hepatitis virus infec-
tion. Antimicrob. Agents Chemother. 44:1964–1969.
8. Hostetler, K. Y., J. R. Beadle, G. D. Kini, M. F. Gardner, K. N. Wright, T.-H.
Wu, and B. A. Korba. 1997. Enhanced oral absorption and antiviral activity
of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir in hepatitis B virus infec-
tion in vitro. Biochem. Pharmacol. 53:1815–1822.
Flanigan, P. N. Kumar, L. Mintz, F. R. Wallach, and G. J. Nemo. 2001.
Highly active antiretroviral therapy decreases mortality and morbidity in
patients with advanced HIV disease. Ann. Intern. Med. 135:17–26.
13. Oliyai, R., J.-P. Shaw, C. M. Sueoke-Lennen, K. C. Cundy, M. N. Arimilli,
R. J. Jones, and W. A. Lee. 1999. Aryl ester prodrugs of cyclic HPMPC I:
physicochemical characterization and in vitro biological stability. Pharm. Res.
16:1687–1693.
14. Qiu, Y.-L., M. B. Ksebati, R. G. Ptak, B. Y. Fan, J. M. Breitenback, J.-S. Lin,
Y.-C. Cheng, E. R. Kern, J. C. Drach, and J. Zemlicka. 1998. (Z)- and
(E)-2-((Hydroxymethyl)cyclopropylidene)methyladenine and -guanosine.
New nucleoside analogs with a broad-spectrum antiviral activity. J. Med.
Chem. 41:10–23.
9. Hostetler, K. Y., R. J. Rybak, J. R. Beadle, C. B. Hartline, M. F. Gardner,
K. A. Aldern, K. N. Wright, and E. R. Kern. 2001. In vitro and in vivo activity
of 1-O-hexadecylpropanediol-3-phospho-ganciclovir and 1-O-hexadecylpro-
panediol-3-phospho-penciclovir in cytomegalovirus and herpes simplex virus
infections. Antivir. Chem. Chemother. 11:373–381.
10. Kern, E. R., C. Hartline, E. Harden, K. Keith, N. Rodriguez, J. R. Beadle,
and K. Y. Hostetler. 2002. Enhanced inhibition of orthopoxvirus replication
in vitro by alkoxylalkyl esters of cidofovir and cyclic cidofovir. Antimicrob.
Agents Chemother. 46:991–995.
15. Stanat, S. C., J. E. Reardon, A. Erice, M. C. Jordan, W. L. Drew, and K. K.
Biron. 1991. Ganciclovir-resistant cytomegalovirus clinical isolates: mode of
resistance to ganciclovir. Antimicrob. Agents Chemother. 35:2191–2197.
16. Sullivan, V., K. K. Biron, C. Talarico, S. C. Stanat, M. Davis, L. M. Pozzi,
and D. M. Coen. 1993. A point mutation in the human cytomegalovirus DNA
polymerase gene confers resistance to ganciclovir and phosphonylmethoxy-
alkyl derivatives. Antimicrob. Agents Chemother. 37:19–25.
17. Sullivan, V., C. L. Talarico, S. C. Stanat, M. Davis, D. M. Coen, and K. K.
Biron. 1992. A protein kinase homologue controls phosphorylation of gan-
ciclovir in human cytomegalovirus-infected cells. Nature 358:162–164.
18. Winston, D. J., D. Wirin, A. Shaked, and R. W. Busuttil. 1995. Randomized
comparison of ganciclovir and high-dose acyclovir for long-term cytomega-
lovirus prophylaxis in liver-transplant recipients. Lancet 346:69–74.
11. Kern, E. R., J. C. Overall, Jr., and L. A. Glasgow. 1973. Herpesvirus hominis
infection in newborn mice. I. An experimental model and therapy with
iododeoxyuridine, J. Infect. Dis. 128:290–299.
12. Murphy, E. L., A. C. Collier, L. A. Kalish, S. F. Assmann, M. F. Para, T. P.