Synthesis and antiviral evaluation of 9-(S)-[3-alkoxy-2-(phosphonomethoxy) propyl]nucleoside alkoxyalkyl esters: Inhibitors of hepatitis C virus and HIV-1 replication

Article abstract of DOI:10.1016/j.bmc.2011.06.009

We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2- (phosphonomethoxy)-propyl]adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This is surprising because acyclic nucleoside phosphonates have be

Full text of DOI:10.1016/j.bmc.2011.06.009

Bioorganic & Medicinal Chemistry 19 (2011) 4616–4625
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiviral evaluation of 9-(S)-[3-alkoxy-2-(phosphonomethoxy)-
propyl]nucleoside alkoxyalkyl esters: Inhibitors of hepatitis C
virus and HIV-1 replication
Nadejda Valiaeva a,b, David L. Wyles a, Robert T. Schooley a, Julia B. Hwu b, James R. Beadle a,b
,
Mark N. Prichard c, Karl Y. Hostetler a,b,
⇑
a Division of Infectious Diseases, University of California, San Diego, United States
b Veterans Medical Research Foundation, La Jolla, CA, United States
c Department of Pediatrics, University of Alabama School of Medicine, Birmingham, AL, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We reported previously that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]
adenine (ODE-(S)-HPMPA) was active against genotype 1b and 2a hepatitis C virus (HCV) replicons. This
is surprising because acyclic nucleoside phosphonates have been regarded as having antiviral activity
only against double stranded DNA viruses, HIV and HBV. We synthesized octadecyloxyethyl 9-(S)-
[3-methoxy-2-(phosphonomethoxy)propyl]-adenine and found it to be active in genotype 1b and 2a
Received 5 April 2011
Revised 27 May 2011
Accepted 2 June 2011
Available online 13 June 2011
HCV replicons with EC50 values of 1–2 lM and a CC50 of >150 lM. Analogs with substitutions at the
Keywords:
Antiviral prodrugs
Hepatitis C virus
Human immunodeficiency virus-1
Alkoxyalkyl prodrugs
Nucleoside phosphonate prodrugs
30-hydroxyl larger than methyl or ethyl, or with other purine bases were less active but most compounds
had significant antiviral activity against HIV-1 in vitro. The most active anti-HIV compound was octade-
cyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]guanine with an EC50 <0.01 nanomolar and
a selectivity index of >4.4 million.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors, NS5A inhibitors3 and NS5B antiviral nucleosides and
non-nucleoside polymerase inhibitors.4 Clinical and in vitro data
Treatment of hepatitis C virus (HCV) infection remains an
important unmet medical need due to the inadequacies of current
interferon-based therapy.1 In the United States there are 3–4 mil-
lion persons with chronic HCV infection.2 A number of agents are
currently in clinical development for HCV including NS3 protease
indicate that resistance develops readily with protease and non-
nucleoside polymerase inhibitors.5–7 In contrast, nucleoside inhib-
itors, which target the catalytic site of the NS5B RNA dependent
RNA polymerase, are active across different HCV genotypes and ex-
hibit a higher barrier to resistance.8
Acyclic nucleoside phosphonates (ANPs) are an important
group of broad spectrum antiviral agents with activity against
double stranded DNA (dsDNA) viruses,9 or viruses which rely on
reverse transcription such as HBV10 and HIV-1.11 We previously
reported that octadecyloxyethyl 9-(S)-[3-hydroxy-2-(phosphono-
methoxy)propyl]adenine (ODE-(S)-HPMPA, 1) exhibited antiviral
activity against genotype 1b and 2a HCV replicons, the first
report of significant antiviral activity of an acyclic nucleoside
phosphonate against an RNA virus.12 However, ODE-(S)-HPMPA
has substantial cytotoxicity.12,13 To identify additional anti-HCV
compounds with less cytotoxicity, we synthesized several closely
related analogs having methyl, ethyl or isopropyl substitutions at
the 30-hydroxyl, octadecyloxyethyl 9-(S)-[3-methoxy-2-(phospho-
nomethoxy)propyl]adenine (ODE-(S)-MPMPA, 15), octadecyloxy-
Abbreviations: (S)-HPMPA, 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-
adenine; (S)-MPMPA, 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]adenine;
ODE, octadecyloxyethyl; HDP, hexadecyloxypropyl; ODE-(S)-MPMPA, octadecyl-
oxyethyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]adenine; ODE-(R)-MPMPA,
octadecyloxyethyl 9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]adenine; HDP-
(S)-MPMPA, hexadecyloxypropyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]
adenine; HDP-(R,S)-EPMPA, hexadecyloxypropyl 9-(R,S)-[3-ethoxy-2-(phosphono-
methoxy)propyl]adenine; HDP-(R,S)-IPPMPA, hexadecyloxypropyl 9-(R,S)-[3-isopro-
poxy-2-(phosphonomethoxy)propyl]adenine; ODE-(S)-MPMPDAP, octadecyloxyethyl
9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]2,6-diaminopurine; HDP-(R,S)-
EPMPDAP, hexadecyloxypropyl 9-(R,S)-[3-ethoxy-2-(phosphonmethoxy)pro-
pyl]2,6-diaminopurine; ODE-(S)-MPMPG, octadecyloxyethyl 9-(S)-[3-methoxy-
2(phosphonomethoxy)propyl]guanine; ODE-(S)-MPMPC, octadecyloxyethyl 1-
(S)-[3-methoxy-2-(phosphonmethoxy)propyl]cytosine; HDP-(S)-MPMPMP, hex-
adecyloxypropyl 9-(S)-[3-methoxy-2-(phosphonomethoxy)propyl]6-methoxypu-
rine; HDP-(S)-MPMPOMG, hexadecyloxypropyl 9-(S)-[3-methoxy-2-(phospho-
nomethoxy)propyl]6-O-methylguanine.
ethyl
9-(R)-[3-methoxy-2-(phosphonomethoxy)propyl]adenine
(ODE-(R)-MPMPA, 16), hexadecyloxypropyl 9-(S)-[3-methoxy-
2-(phosphono-methoxy)propyl]adenine (HDP-(S)-MPMPA, 17),
⇑
Corresponding author.
E-mail address: khostetler@ucsd.edu (K.Y. Hostetler).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.009

N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
4617
NH2
N
NHMMTr
N
NH2
N
N
N
N
N
N
a
b
c
N
N
N
N
H
OH
O
OH
O
R1
R1
R1
=
R1 =
6
2
3
4
5
Me (S)
Me (R)
Et (R,S)
Me (S)
7
8
9
Me (R)
Et (R,S)
iPr (R,S)
iPr (R,S)
NH2
NHMMTr
N
N
N
N
N
d
O
P
O
P
N
O
R2
N
N
O
R2
′
O
1
O- Na+
O
O- Na+
′
2
′
3
O
R1
O
R1
10
15
16
17
18
19
R1 = Me (S) R2 = ODE
R1 = Me (R) R2 = ODE
R1 = Me (S) R2 = HDP
R1 = Et (R,S) R2 = HDP
R1 = iPr (R,S) R2 = HDP
R1 = Me (S) R2 = ODE
R1 = Me (R) R2 = ODE
R1 = Me (S) R2 = HDP
R1 = Et (R,S) R2 = HDP
R1 = iPr (R,S) R2 = HDP
11
12
13
14
HDP
ODE
= -(CH2)3O(CH2)15CH3;
= -(CH2)2O(CH2)17CH3
Scheme 1. Synthesis of adenine derivatives (15–19). Reagents and conditions: (a) NaH, alkyl glycidyl ether, N,N-DMF, 100 °C, 6 h; (b) TMSBr, MMTrCl, pyridine; (c) sodium t-
butoxide, hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) p-toluenesulfonyloxymethylphosphonate, N,N-DMF, 80 °C, 16 h; (d) 80% aq acetic acid, 60 °C, 2 h.
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
N
a
b
O
P
H2N
H2N
O
O- Na+
R2
N
H
OH
O
H2N
O R1
O R1
R1 =
23 R1 = Me (S); R2 = ODE
24 R1 = Me (R); R2 = ODE
20 Me (S)
21
Me (R)
22 Et (R,S)
25
26
R1 = Me (S); R2 = HDP
R1 = Et (R,S); R2 = HDP
HDP
ODE
= -(CH2)2O(CH2)17CH3
= -(CH2)3O(CH2)15CH3;
Scheme 2. Synthesis of 2,6-diaminopurine derivatives (23–26). Reagents and Conditions: (a) NaH, alkyl glycidyl ether, N,N-DMF, 100 °C, 6 h; (b) sodium t-butoxide,
alkoxyalkyl p-toluenesulfonyloxymethylphosphonate, N,N-DMF, 80 °C.
OBn
N
OBn
N
OBn
N
O
N
N
N
N
N
N
N
N
N
N
N
HN
H2N
a
b
c
O
P
O
P
N
H
H2N
H2N
H2N
O R2
O- Na+
O R2
O- Na+
OH
O
O
O R1
O R1
O R1
R2 =
Me (S) ODE
R1 =
R1 =
R2 =
R1 =
27
30
34
Me (S)
28 Me (R)
29
Me (S) ODE
31 Me (R) ODE
35 Me (R) ODE
32
36
Me (S) HDP
37 Et (R,S) HDP
Et (R,S)
Me (S) HDP
33 Et (R,S) HDP
HDP
ODE
= -(CH2)2O(CH2)17CH3
= -(CH2)3O(CH2)15CH3;
Scheme 3. Synthesis of guanine derivatives (34–37). Reagents and conditions: (a) NaH, alkyl glycidyl ether, N,N-DMF, 100 °C, 6 h; (b) sodium t-butoxide, hexadecyloxypropyl
(HDP) or octadecyloxyethyl (ODE) p-toluenesulfonyloxymethylphosphonate, N,N-DMF, 80 °C; (c) 10% CF3COOH/CH2Cl2, rt, two days.

4618
N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
NHMMTr
NHMMTr
NHMMTr
NH2
N
N
N
N
N
a
b
c
O
P
O
P
O
N
H
O
N
O
N
O
O
O ODE
O- Na+
O ODE
O- Na+
OH
O
OCH3
OCH3
39
OCH3
40
38
Scheme 4. Synthesis of cytosine derivative (40). Reagents and conditions: (a) NaH, (S)-methyl glycidyl ether, N,N-DMF, 100 °C, 6 h; (b) octadecyloxyethyl (ODE) p-
toluenesulfonyloxymethylphosphonate; (c) 80% aq acetic acid, 60 °C, 2 h.
OCH3
N
OCH3
OCH3
N
N
N
N
N
N
N
N
a
b
O
P
N
H
N
R
R
N
R
HDP
O
OH
O
O- Na+
OCH3
OCH3
R =
H
NH2
R =
H
NH2
41
42
43
44
Scheme 5. Synthesis of 6-methoxypurine derivatives (43–44). Reagents and conditions: (a) NaH, (S)-methyl glycidyl ether, N,N-DMF, 100 °C, 6 h; (b) sodium t-butoxide,
hexadecyloxypropyl (HDP) p-toluenesulfonyloxymethylphosphonate, N,N-DMF, 80 °C.
hexadecyloxypropyl
9-(R,S)-[3-ethoxy-2-(phosphonomethoxy)-
EC50 values of 1.43 0.38 and 2.38 1.09
(16) was slightly less active with EC50 of 4.65 and 5.33
HDP-(S)-MPMPA (17) was slightly less active than the correspond-
l
M while the (R) isomer
propyl]adenine (HDP-(R,S)-EPMPA, 18) and hexadecyloxypropyl
9-(R,S)-[3-isopropoxy-2-(phosphonomethoxy)propyl]-adenine
(HDP-(R,S)-IPPMPA, 19) (Scheme 1). We also prepared the HDP-
or ODE- esters of (R and S)-MPMP-2,6-diaminopurine (23–26)
(Scheme 2), guanine (34–37) (Scheme 3), cytosine (40), 6-meth-
oxypurine (43) (Scheme 4) and 6-O-methylguanine (44) (Scheme
5). The antiviral activity of these compounds against HCV, HIV-1
and other viruses was compared with ODE-(S)-HPMPA (1).
lM.
ing ODE ester with EC50 of 2.36 (1b) and 4.64 lM (2a). When ethyl
or isopropyl substitutions were made at the 30-hydroxyl instead of
methyl, the anti-HCV activity dropped slightly with HDP-(R,S)-
EPMPA (18) to EC50 of 7.59 (1b) and 8.87 lM (2a) suggesting that
larger substitutions are not favored. Cytotoxicity of ODE-(S)-
MPMPA was substantially lower than that observed with
ODE-(S)-HPMPA, CC50 >150 versus 35.6 lM. Of the various adenine
analogs, ODE-(S)-MPMPA had the greatest selectivity index, >105
with genotype 1b and >63 with genotype 2a replicons. The
anti-HCV activity of ODE-(S)-MPMPA is similar to that of
2. Results and discussion
2.1. Chemistry
40-azidocytidine and 20-C-methylcytidine, EC50 1.13–1.28 M,15
l
and less than that of PSI-352938, a cyclic phosphate prodrug of
b-
-20- -fluoro-20-b-C-methylguanosine, EC50 0.13–0.20 M.16
We also prepared alkoxyalkyl MPMP esters of cytosine, guanine,
2,6-diaminopurine, 6-methoxypurine and 6-O-methylguanine.
Among them, the most active anti-HCV compound was ODE-(S)-
Synthesis of the new ANP analogs was carried out using the
synthon approach we developed previously for alkoxyalkyl esters
D
a
l
of
(S)-9-[3-hydroxy-2-(phosphonomethoxy)-propyl]adenine
[(S)-HPMPA].13 As detailed in Scheme 1, adenine reacted with
various alkyl glycidyl ethers under basic conditions to give a series
of 9-(3-alkoxy-2-hydroxypropyl)adenines (2–5). After protection
of the amino group with monomethoxytrityl, compounds 6–9
could be alkylated with octadecyloxyethyl or hexadecyloxypropyl
p-toluenesulfonyloxymethyl-phosphonate, then deprotected to
provide adenine derivatives 15–19. As shown in Scheme 2, 3-
methoxy- and 3-ethoxy-2-phosphonomethoxypropyl analogs of
2,6-diaminopurine (23–26) were prepared using a similar strategy.
In this case, acceptable yields (22–50%) of the target compounds
were obtained without protection of the amino groups. Synthesis
of the related guanine (Scheme 3 and 34–37) and cytosine (Scheme
MPMPG (34) with EC50 values of 8.26 and 10.7
type 1b and 2a, respectively; the (R) isomer (35) was slightly less
active with EC50 of 12.6 and 12.4 M. These compounds also had
low cytotoxicity with CC50 values >150 M. HDP-(S)-MPMPMP
(43) also exhibited significant activity in the 8.9–12.5 M range.
lM against geno-
l
l
l
HDP-(S)-MPMPOMG (44) and the ODE and HDP esters of both (R)
and (S)-MPMPDAP (23–25) were less active with EC50 values rang-
ing from 18 to 26 lM while ODE-(S)-MPMPC (40) was inactive.
We also evaluated the compounds in MT-2 cells infected with
HIV-1 (Table 2). ODE-(S)-HPMPA (1) was highly active with an
4
and 40) analogs started from 6-O-benzylguanine and N4-
EC50 of 0.0001
the most cytotoxic compound in the series. ODE-(S)-MPMPA (15)
retained substantial antiviral activity with an EC50 of 0.03
and a CC50 of 22 M (selectivity index 733). By way of comparison,
lM. However, the CC50 was 0.033 lM making it
monomethoxytritylcytosine, respectively. Methoxypurine analogs
(43–44) were prepared as shown in Scheme 5.
lM
l
our previous studies with adefovir and tenofovir in HIV-1 infected
2.2. Biological activity
MT-2 cells showed EC50 values for these compounds of 1.1 and
0.65 l
M, respectively.17,18 HDP-(S)-MPMPA (17) was less active
Compounds were tested for anti-HCV activity in genotype 1b
and 2a replicons as previously described14 and their activity com-
pared with that of ODE-(S)-HPMPA (Table 1). ODE-(S)-MPMPA (15)
retained full activity against genotype 1b and 2a replicons with
and ODE-(R)-MPMPA (16) was considerably less active and selec-
tive. Introduction of an ethoxy or isopropoxy at the 30-hydroxyl po-
sition of the acyclic moiety (compounds 18, 19) resulted in a loss of

N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
4619
Table 1
HCV replicon activity and cytotoxicity
O
B
O
P
O
R2
O- Na+
R1
O
Compd
Base
R1
R2
Abbreviation
EC50
(
l
M)
CC50
(
lM)
Selectivity index
BM4–5 (1b)
Selectivity
index JFH-1 (2a)
BM4–5(1b)
JFH-1 (2a)
1
Adenine
H
Me
Me
Me
Ethyl
Isopropyl
Me
Me
Me
Ethyl
Me
Me
Me
Ethyl
Me
ODE
ODE
ODE
HDP
HDP
HDP
ODE
ODE
HDP
HDP
ODE
ODE
HDP
HDP
ODE
HDP
HDP
ODE-(S)-HPMPA
ODE-(S)-MPMPA
ODE-(R)-MPMPA
HDP-(S)-MPMPA
HDP-(R,S)-EPMPA
HDP-(R,S)-IPPMPA
ODE-(S)-MPMPDAP
ODE-(R)-MPMPDAP
HDP-(S)-MPMPDAP
HDP-(R,S)-EMPDAP
ODE-(S)-MPMPG
ODE-(R)-MPMPG
HDP-(S)-MPMPG
HDP-(R,S)-EPMPG
ODE-(S)-MPMPC
HDP-(S)-MPMPMP
1.55 0.50
1.43 0.38
4.65 0.88
2.36 0.37
7.59 1.31
51.2 38.2
20.1 1.98
25.6 4.23
21.3 3.10
18.2 7.09
8.26 1.30
12.6 1.67
22.0 4.97
25.5 10.4
>150
1.65 0.33
2.38 1.09
5.33 0.92
4.64 1.26
8.87 2.05
98.8 20.8
21.4 1.30
25.8 6.10
23.6 2.70
19.5 1.48
10.7 1.33
12.4 3.45
24.8 7.00
13.2 3.45
>150
35.6 6.8
>150
>150
22.9
>105
>32.3
>63.6
13.0
3.14
4.94
>5.86
4.30
>8.2
>18.2
>11.9
>6.82
>5.88
—
21.6
>63
>28.1
>32.3
11.1
—
4.62
>5.81
3.87
>7.69
>14.0
>12.1
>6.04
>11.4
—
15
16
17
18
19
23
24
25
26
34
35
36
37
40
43
44
>150
99 0.5
100 20.8
99.0 13.4
>150
91.5 17.8
>150
>150
>150
>150
>150
2,6-Diamino-purine
Guanine
Cytosine
6-Methoxy-purine
6-O-Methyl-guanine
>150
>150
>150
Me
Me
8.90 1.22
18.1 0.27
12.5 3.36
17.4 0.30
8.04
4.98
5.72
5.18
HDP-(S)-MPMPOMG
Data are mean standard deviation (n = 3).
antiviral activity (Table 2). Surprisingly, the most active compound
lM and a selectivity
index of >4.4 million. Interestingly, the (S) isomer (34) was
was ODE-(R)-MPMPG (35), EC50 <1 ꢀ 10ꢁ5
Table 2
Antiviral activity against HIV-1 and cytotoxicity in MT-2 cells
substantially less active with an EC50 of 0.2
was observed with ODE-(R)-MPMPDAP (24) which was more
active (EC50 = 0.4 M) than the (S) isomer (23). As noted before
with the adenine analogs, the HDP esters (25, 36) were less active.
Again, introduction of larger ethyl groups at the 30-hydroxyl of the
acyclic chain of these compounds (26, 37) caused a large loss of
anti-HIV activity. ODE-(S)-MPMPC (40) was less potent
lM. The same pattern
Compound EC50 M) CC50 M)
(
l
(
l
Selectivity
index
l
1
ODE-(S)-HPMPA
0.0001 0.000
(6)
0.033 0.02 (3) 330
15 ODE-(S)-MPMPA
16 ODE-(R)-MPMPA
17 HDP-(S)-MPMPA
18 HDP-(R,S)-EPMPA
19 HDP-(R,S)-IPPMPA
23 ODE-(S)-MPMPDAP
24 ODE-(R)-MPMPDAP 0.04 0.06 (4)
25 HDP-(S)-MPMPDAP
26 HDP-(R,S)-
0.03 0.015 (3)
4.8 3.0 (3)
0.20 0.26 (4)
>10
>10
0.23 0.15
22 5 (3)
26.7 11.2 (3) 5.6
733
32 11 (3)
23.6 7.1 (3)
30.3 12.5 (3)
18.0 5.3 (3)
19.7 9.5 (3)
22.7 6.8 (3)
30.7 11.7 (3)
160
—
—
78
493
4.9
—
(EC50 = 12.7
POMG (44) were considerably less potent against HIV (EC50
10 M).
ODE-(S)-MPMPA was also tested against HCMV and HSV-1
lM) and HDP-(S)-MPMPMP (43) and HDP-(S)-MPM-
>
l
4.6 1.8 (3)
>10
using our previous methods.19 We reported previously that ODE-
(S)-HPMPA is a powerful inhibitor of the replication of orthopoxvi-
ruses, including variola,20 vaccinia and cowpox,21 and ectromelia,22
as well as other dsDNA viruses including human cytomegalovirus
(HCMV) and herpes simplex virus, type 1 (HSV-1).13 We examined
the effect of blocking the 30-hydroxyl of HPMPA with 30-methoxy
on the compound’s antiviral activity against dsDNA viruses includ-
ing vaccinia, cowpox, HCMV and HSV-1 (Table 3). As we reported
previously, ODE-(S)-HPMPA had potent antiviral activity against
these viruses with EC50 values ranging from <0.1 to 20 nanomolar.
However, ODE-(S)-MPMPA (15) exhibited a dramatic loss of antivi-
ral activity with EC50 values >400 to >45 million times higher than
EPMPDAP
34 ODE-(S)-MPMPG
35 ODE-(R)-MPMPG
36 HDP-(S)-MPMPG
37 HDP-(R,S)-EPMPG
40 ODE-(S)-MPMPC
43 HDP-(S)-MPMPMP
44 HDP-(S)-
0.20 0.22 (4)
<1 ꢀ 10ꢁ5 (3)
2.03 0.95 (4)
>10
12.7 4.0 (3)
>10
25 13 (3)
44 5.6 (3)
125
>4.4 ꢀ 106
29.3 3.1 (3)
14.9 6.7 (3)
60.7 16 (3)
22.0 3.5 (3)
34.3 7.6 (3)
14.4
—
4.8
—
>10
—
MPMPOMG
Antiviral and cytotoxicity methods as described previously (Ref. 11). Data are
mean std. deviation. The number of replicates is in parentheses.
Table 3
Comparative antiviral activity of ODE-(S)-HPMPA versus ODE-(S)-MPMPA against dsDNA viruses in vitro
Compound
EC50 (lM)
Vaccinia
Cowpox
HCMV
HSV-1
1
15
ODE-(S)-HPMPA
ODE-(S)-MPMPA
0.02 0.01a
18.3 2.4
915
0.05 0.04a
>20.0
>400
0.003 0.001a
1.55 0.4b
516
<0.0001
45.7 10.1b
>45 million
Fold change
a
Data and methods from Beadle et al.(Ref. 13).
Data for ODE-(S)-MPMPA versus HCMV and HSV-1 were obtained by plaque reduction assay in HFF cells as previously described by Prichard et al.(Ref. 19).
b

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N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
those of ODE-(S)-HPMPA (Table 3). We reported previously that
inhibition of vaccinia virus replication occurs by a unique mecha-
nism in which (S)-HPMPA diphosphate is incorporated into DNA
by the viral E9L polymerase. However, the vaccinia polymerase
cannot copy across the drug lesion in HPMPA containing tem-
plates.23 The EC50 for vaccinia inhibition by ODE-(S)-HPMPA is
20 nanomolar versus 18,300 nanomolar for ODE-(S)-MPMPA, a
reduction of 915-fold (Table 3). These findings generally support
the principal mechanism which we described previously because
incorporation of HPMPA into viral DNA, blocking further copying
of the drug-containing chain is not possible with ODE-(S)-MPMPA,
and any residual antiviral activity with the latter compound is due
to obligatory chain termination. Presumably this is also the
mechanism of action in cowpox which has a closely related DNA
polymerase. The antiviral mechanism of action of HPMPA diphos-
phate has not been studied extensively for HCMV and HSV-1.
(20–25 min.); 95% to 10% B (25–34 min.). Compounds were de-
tected by ultraviolet light (UV) absorption at 274 nm. Purity of
compounds was also assessed by thin layer chromatography
(TLC) using Analtech silica gel-GF (250 lm) plates and the solvent
system: CHCl3/MeOH/conc NH4OH/H2O (70:30:3:3 v/v). TLC
results were visualized with UV light, phospray (Supelco,
Bellefonte, PA, USA) and charring at 400 °C. The key synthons,
hexadecyloxypropyl- and octadecyloxyethyl p-toluenesulfonylo-
xymethylphosphonate, were prepared as described previously.13
4.2. General procedure A. Synthesis of 3-alkoxy-2-hydroxy-
propyl nucleoside analogs
The preparation of various 3-alkoxy-2-hydroxypropyl nucleo-
side analogs was accomplished using the base catalyzed ring-open-
ing reaction of alkyl glycidyl ethers with nucleobases as described
by Brodfuehrer et al.24 Sodium hydride (2 mmol) was added to a
solution of the nucleic acid base (10 mmol) and an alkyl glycidyl
ether (10 mmol) in anhydrous N,N-dimethylformamide (50 mL)
and the mixture was stirred and heated to 100 °C for 6 h. After
cooling, the reaction was quenched with H2O, the solvent was re-
moved in vacuo and the residue was purified by flash column chro-
matography on silica gel. Elution of the column with 10% MeOH/
CH2Cl2 gave the product.
3. Conclusions
Prior studies identified ODE-(S)-HPMPA (1) as a compound with
good antiviral activity against HCV.12 However, this compound has
significant toxicity both in vitro and in mice, in vivo. Surprisingly,
replacement of the acyclic chain hydroxyl with a methoxy group
greatly reduces toxicity while anti-HCV activity is preserved in
the 1–2 lM range. Selectivity indexes for ODE-(S)-MPMPA (15)
4.2.1. (S)-9-[(3-Methoxy-2-hydroxy)propyl]adenine (2)
Compound 2 was synthesized from adenine and (S)-methyl
glycidyl ether (TCI America, Portland, OR). 75% yield. 1H NMR
(CDCl3/methanol-d4) d 8.25 (s, 1H); 8.03 (s, 1H); 4.39–4,44 (m,
1H); 4.19–4.25 (m, 1H); 4.08–4.18 (m, 1H); 3.37–3.44 (m, 2H);
3.40 (s, 3H). MS (ESI): 224.14 [M+H]+.
range from >63 to >105, a marked improvement over the selectiv-
ity of ODE-(S)-HPMPA (1). Interestingly, blocking the 30-hydroxyl
of ODE-(S)-HPMPA with a methyl results in a marked loss of anti-
viral activity against double stranded DNA viruses, including vac-
cinia, cowpox, HCMV and HSV-1. The ODE-MPMP- analogs of
guanine, 6-methoxypurine and 6-O-methylguanine were also ac-
tive against HCV while the ODE (S) and (R) isomers of MPMPDAP
were somewhat less potent. ODE-(S)-MPMPC was inactive.
ODE-(R)-MPMPG (35) showed striking antiviral activity against
HIV-1 with an EC50 of <0.01 nanomolar and a selectivity index of
>4.4 million. Our results support further evaluation of ODE-(S)-
MPMPA (15) for use against chronic HCV infection and ODE-(R)-
MPMPG (35) for HIV infection.
4.2.2. (R)-9-[(3-Methoxy-2-hydroxy)propyl]adenine (3)
Compound 3 was synthesized from adenine and (R)-methyl
glycidyl ether (TCI America, Portland, OR). 72% yield. 1H NMR
(methanol-d4) d 8.20 (s, 1H, H-8); 8.08 (s, 1H, H-2); 4.39 (dd, 1H,
H-10a, J1 a,2 = 3.4 Hz, Jgem = 14.2 Hz); 4.20 (dd, 1H, H-10b,
0
0
J1 b,2 = 8.0 Hz, Jgem = 14.2 Hz); 4.11 (m, 1H, H-20); 3.42 (d, 2H,
0
0
H-30, J3 ,2 = 5.2 Hz); 3.37 (s, 3H, –OCH3).
0
0
4. Experimental
4.2.3. (R,S)-9-[(3-Ethoxy-2-hydroxy)propyl]adenine (4)
Compound 4 was synthesized from adenine and (R,S)-ethyl
glycidyl ether (TCI America) with 59% yield. 1H NMR (CDCl3/meth-
anol-d4), d 8.24 (s, 1H); 8.04 (s, 1H); 4.40–4.65 (m, 1H); 4.20–4.27
(m, 1H); 4.10–4.15 (m, 1H); 3.55–3.57 (m, 2H); 3.46–3.54 (m, 2H);
1.21 (t, J=7 Hz, 3H). MS (ESI): 238.09 [M+H]+.
4.1. General chemistry methods
All reagents were of commercial quality and used without fur-
ther purification unless indicated otherwise. Chromatographic
purification was done using the flash method with silica gel 60
(EMD Chemicals, Inc., 230–400 mesh). 1H NMR spectra were re-
corded on Varian HG spectrophotometers operating at 400 MHz
and are reported in units of parts per million (ppm) relative to
internal tetramethylsilane at 0.00 ppm. Assignments of 1H NMR
signals are made using the numbering system shown in Scheme
1. Routine electrospray ionization mass spectra (ESI-MS) were re-
corded on a Finnigan LCQDECA spectrometer, and high resolution
mass spectra (HRMS) on an Agilent 6230 Accurate-Mass TOFMS
mass spectrometer in ESI negative mode. Both spectrometers are
located at the small molecule facility, Department of Chemistry,
University of California, San Diego and operated by Dr. Yongxuan
Su. Purity of the target compounds was characterized by high per-
formance liquid chromatography (HPLC) using a Beckman Coulter
System Gold chromatography system. The analytical column was
Phenomenex Synergi™ Polar-RP (4.6 ꢀ 150 mm) equipped with a
SecurityGuard™ protection column. Mobile phase A was 95%
water/5% methanol and mobile phase B was 95% methanol/5%
water. At a flow rate of 0.8 mL/min, gradient elution was as
4.2.4. (R,S)-9-[(3-Isopropoxy-2-hydroxy)propyl]adenine (5)
Compound 5 was synthesized from adenine and (R,S) isopropyl
glycidyl ether (Aldrich Chem.). 33.8% yield. 1H NMR (CDCl3/metha-
nol-d4), d 8.21 (s, 1H, H-8); 8.08 (s, 1H, H-2); 4.41 (dd, 1H, H-10a,
J1 a,2 = 3.8, Jgem = 14.2 Hz); 4.23 (dd, 1H, H-10b, J1 b,2 = 7.6 Hz,
Jgem = 14.4 Hz); 4.10 (m, 1H, H-20); 3.60 (septet, 1H, –CH(CH3)2,
J = 6.0 Hz); 1.16 (d, 6H, –CH(CH3)2).
0
0
0
0
4.3. General procedure B. Synthesis of 9-[(3-alkoxy-2-hydroxy)-
propyl]-N6-monomethoxytrityladenine (6–9)
The monomethoxytrityl group was used to block the exocylic
amino group of adenine and was introduced by the transient pro-
tection method described by Ti et al.25 Bromotrimethylsilane
(6.3 mmol) was added dropwise to a suspension of 9-[(2-hydro-
xy-3-alkoxy)propyl]adenine (2–5) (2.8 mmol) in dry pyridine
(10 mL). The mixture was stirred 15 min. until it became clear,
then monomethoxytrityl chloride (0.99 g, 3.2 mmol) and
4-(dimethylamino)pyridine (20 mg, 0.2 mmol) were added and
follows: 10%
B (0–3 min.); 10–95% B (3–20 min.); 95% B

N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
4621
stirring was continued overnight. The mixture was cooled with an
ice bath and H2O (1 mL) was added. Stirring was continued
10 min., then con. NH4OH (1 mL) was added and the reaction
was stirred 30 additional min. The mixture was allowed to warm
to room temperature and filtered through a pad of celite. The
filtrate was evaporated in vacuo and the residue was purified by
flash column chromatography on silica gel. Gradient elution
(100% hexanes to 100% ethyl acetate) afforded the N6-monometh-
oxytrityl 9-[(2-alkoxy-3-methoxy)propyl]adenines (6–9).
cyloxyethyl p-toluenesulfonyloxymethylphosphonate. 60% yield.
1H NMR (CDCl3/methanol-d4), d 8.18 (s, 1H); 7.98 (s, 1H); 7.22–
7.55 (m, 14H); 4.38–4.50 (m, 2H); 4.12–4.37 (m, 2H); 4.00–4.08
(m, 1H); 3.82–3.98 (m, 2H); 3.79 (s, 3H); 3.58–3.65 (m, 2H);
3.44–3.48 (m, 2H); 3.38–3.43 (m, 2H); 3.35 (s, 3H); 1.40–1.60 (m,
2H); 1.16–1.38 (m, 30H); 0.88 (t, J = 7 Hz, 3H). MS (ESI): 886.48
[M+H]+.
4.4.2. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphono-
methoxy)propyl] N6-monomethoxytrityladenine, sodium salt
(11)
4.3.1. (S)-9-[(3-Methoxy-2-hydroxy)propyl]-N6-monomethoxy-
trityladenine (6)
Compound 11 was synthesized from (R)-9-[(3-methoxy-2-
hydroxy)propyl]-N6-monomethoxytrityladenine (7) and octade-
cyloxyethyl p-toluenesulfonyloxymethylphosphonate. 43% yield.
1H NMR (CDCl3/methanol-d4), d 8.20 (s, 1H); 7.98 (s, 1H); 7.22–
7.37 (m, 14H); 4.42–4.50 (m, 1H); 4.28–4.37 (m, 1H); 3.91–3.98
(m, 2H); 3.82–3.90 (m, 2H); 3.79 (s, 3H); 3.60–3.69 (m, 1H);
3.48–3.58 (m, 3H); 3.39–3.46 (m, 2H); 3.35 (s, 3H); 1.45–1.60 (m,
2H); 1.20–1.38 (m, 30H); 0.88 (t, J=7 Hz, 3H). MS (ESI): 886.57
[M+H]+.
Compound 6 was synthesized from 2. 98% yield. 1H NMR
(CDCl3/methanol-d4), d 8.17 (s, 1H, H-8); 8.13 (s, 1H, H-2); 7.39–
7.75 (m, 14H, trityl); 4.50–4.59 (m, 1H); 4.31–4.39 (m, 1H);
4.22–4.30 (m, 1H); 3.45 (s, 3H); 3.50–3.60 (m, 2H); 3.55 (s, 3H).
MS (ESI): 496.06 [M+H]+, 518.13 [M+Na]+.
4.3.2. (R)-9-[(3-Methoxy-2-hydroxy)propyl] N6-monomethoxy-
trityladenine (7)
Compound 7 was synthesized from 3. 19% yield. 1H NMR
(CDCl3/methanol-d4), d 8.03 (s, 1H, H-8); 7.78 (s, 1H, H-2);
7.35–7.33 (m, 4H, trityl); 7.29–7.24 (m, 10H, trityl); 4.61 (d, 1H,
4.4.3. Hexadecyloxypropyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl] N6-monomethoxytrityladenine, sodium salt
(12)
H-10a, J1 a,2 = 4 Hz); 4.39 (dd, 1H, H-10b, J1 b,2 = 2.2 Hz,
0
0
0
0
Jgem = 13.8 Hz); 4.16 (m, 1H, H-20); 3.78 (s, 3H, Ar–OCH3); 3.41
Compound 12 was synthesized from (S)-9-[(3-methoxy-2-
hydroxy)propyl]-N6-monomethoxytrityladenine (6) and hexade-
cyloxypropyl p-toluenesulfonyloxymethylphosphonate. 77% yield.
1H NMR (CDCl3/methanol-d4), d 8.17 (s, 1H); 7.94 (s, 1H); 7.22–
7.36 (m, 14H); 4.38–4.50 (m, 2H); 4.28–4.37 (m, 2H); 3.82–3.98
(m, 2H); 3.79 (s, 3H); 3.58–3.65 (m, 1H); 3.38–3.58 (m, 6H); 3.34
(s, 3H); 1.78–1.87 (m, 2H); 1.44–1.60 (m, 2H); 1.10–1.40 (m,
26H); 0.88 (t, J=7 Hz, 3H). MS (ESI): 870.33 [MꢁH]ꢁ.
(dd, 1H, H-30a, J3 a,2 = 5.2 Hz, Jgem = 9.2 Hz); 3.36 (s, 3H, CH2–
0
0
OCH3); 3.33 (dd, 1H, H-30b, J3 b,2 = 6.2 Hz, Jgem = 9.8 Hz).
0
0
4.3.3. (R,S)-9-[(3-Ethoxy-2-hydroxy)propyl] N6-monomethoxy-
trityladenine (8)
Compound 8 was synthesized from 4. 78% yield. 1H NMR
(CDCl3/methanol-d4), d 7.98 (s, 1H); 7.25–7.34 (m, 14H); 6.82 (s,
1H); 4.35–4.43 (m, 1H); 4.15–4.24 (m, 1H); 4.05–4.15 (m, 1H);
3.78 (s, 3H); 3.52–3.56 (m, 2H); 3.44–3.47 (m, 2H); 1.20 (t,
J=7 Hz, 3H). MS (ESI): 509.78 [M+H]+.
4.4.4. Hexadecyloxypropyl (R,S)-9-[(3-ethoxy-2-phosphono-
methoxy)propyl] N6-monomethoxytrityladenine, sodium salt
(13)
4.3.4. (R,S)-9-[(3-Isopropoxy-2-hydroxy)propyl] N6-mono-
methoxytrityladenine (9)
Compound 13 was synthesized from (R,S)-9-[(3-ethoxy-2-
hydroxy)propyl]-N6-monomethoxytrityladenine (8) and hexade-
cyloxypropyl p-toluenesulfonyloxymethylphosphonate. 80% yield.
1H NMR (CDCl3/methanol-d4), d 8.19 (s, 1H); 7.98 (s, 1H); 7.20–
7.36 (m, 14H); 4.42–4.65 (m, 1H); 4.28–4.37 (m, 1H); 3.80- 3.95
(m, 3H); 3.78 (s, 3H); 3.48–3.65 (m, 6H); 3.28–3.48 (m, 2H);
1.78–1.87 (m, 2H); 1.44–1.55 (m, 2H); 1.08–1.30 (m, 26H); 1.15
(t, J=7 Hz, 3H); 0.88 (t, J=7 Hz, 3H). MS (EI): 886.42 (M+H)+.
Compound 9 was synthesized from 5. 32% yield. 1H NMR
(DMSO-d6) d 8.10 (s, 1H, H-8); 7.89 (s,1H, H-2); 7.28–7.26 (m,
10H, trityl); 7.21–7.18 (m, 4H, trityl); 6.83 (d, 1H, –NH–); 5.16
(d, 1H, –OH); 4.23 (dd, 1H, H-10a, J1 a,2 = 3.4 Hz, Jgem = 13.8 Hz);
0
0
3.98 (dd, 1H, H-10b, J1 b,2 = 8.4 Hz, Jgem = 13.6 Hz); 3.92 (m, 1H, H-
20); 3.70 (s, 3H, Ar–OCH3); 3.50 (septet, 1H, –CH(CH3)2); 3.35 (dd,
0
0
1H, J3 a,2 = 4.8 Hz, Jgem = 10 Hz); 3.27 (dd, 1H, H-30b, J3 b,2 = 6,
Jgem = 9.6); 1.04 (dd, 6H, –CH(CH3)2).
0
0
0
0
4.4.5. Hexadecyloxypropyl (R,S)-9-[(3-isopropoxy-2-
phosphonomethoxy)propyl] N6-monomethoxytrityladenine,
sodium salt (13)
4.4. General procedure C. Alkylation of 9-[(3-alkoxy-2-hydroxy)
propyl] derivatives (6–9, 20–22, 27–29, 38, 41–42) with
alkoxyalkyl p-toluenesulfonyloxymethylphosphonate.
Synthesis of (10–14, 23–26, 30–33, 39, 43–44)
Compound 13 was synthesized from (R,S)-9-[(3-isopropoxy-2-
hydroxy)propyl]-N6-monomethoxytrityladenine (9) and hexade-
cyloxypropyl p-toluenesulfonyloxymethylphosphonate. 25% yield.
1H NMR (CDCl3/methanol-d4) d 8.15 (s, 1H, H-8); 7.88 (s, 1H,
H-2); 7.30–7.25 (m, 4H, trityl); 7.20–7.12 (m, 10H, trityl); 4.66
Sodium t-butoxide (0.19 g, 2.0 mmol) was added to a solution of
the 3-alkoxy-2-hydroxypropyl nucleoside (1.0 mmol) and the al-
koxyalkyl p-toluenesulfonyloxymethylphosphonate12 (2.0 mmol)
in anhydrous N,N-DMF (20 mL). The mixture was heated to 80 °C
and kept overnight. After cooling, the solvents were evaporated
in vacuo and the residue was purified by flash column chromatog-
raphy on silica gel. The column was eluted with a gradient: chloro-
form 100%—chloroform–methanol (20%) to give the products.
(dd, 1H, H-10a, J1 a,2 = 3.5 Hz, Jgem = 14.2 Hz); 4.47 (dd, 1H, H-10b,
0
0
0
0
J1 b2 = 6.2 Hz, Jgem = 14.0 Hz); 4.01 (m, 2H, –P–O–CH2–); 3.88 (dd,
1H, –CHa–P–, JP,CHa = 9.2 Hz, Jgem = 13.6 Hz); 3.71 (dd, 1H, –CHb–
P–, JP,CHb = 9.6 Hz, Jgem = 14.0 Hz); 3.75–3.55 (m, 3H, H-30 + H-20);
3.51 (t, 2H, –CH2–O–CH2–); 3.45 (t, 2H, –CH2–O–CH2–); 1.83 (pen-
tet, 2H, –O–CH2CH2CH2O–); 1.53 (m, 2H, –CH2(CH2)15–); 1.27 (m,
26H, –(CH2)15–); 1.10 (d, 6H, –CH(CH3)2); 0.89 (t, 3H, –CH3).
4.4.1. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl] N6-monomethoxytrityladenine, sodium salt
(10)
4.5. General procedure D. Synthesis of alkoxyalkyl-9-[(3-alkoxy-
2-phosphonomethoxy)propyl]adenine (15–19)
Compound 10 was synthesized from (S)-9-[(3-methoxy-2-
Alkoxyalkyl
oxytrityladenine (10–14) (0.60 mmol) was added to 80% acetic
9-[(3-alkoxy-2-hydroxy)propyl]-N6-monometh-
hydroxy)propyl]-N6-monomethoxytrityladenine (6) and octade-

4622
N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
acid, stirred and heated to 60 °C for 2 h. After cooling, the solvent
was removed in vacuo and the residue purified by flash column
chromatography on silica gel. Elution with 20% MeOH/CH2Cl2 gave
the products.
(E = 0.2 ppm). HPLC analysis: retention time 21.95 min., purity
97.1%.
4.6. 2,6-Diaminopurine derivatives
4.6.1. (S)-9-[(3-Methoxy-2-hydroxy)propyl]-2,6-diaminopurine
(20)
Compound 20 was synthesized from 2,6-diaminopurine (TCI
America) and (S)-methyl glycidyl ether (procedure A). 27% yield
(0.65 g). 1H NMR (CDCl3/methanol-d4), d 7.68 (s, 1H, H-8); 4.20–
4.30 (m, 1H); 4.05–4.12 (m, 2H); 3.32–3.47 (m, 2H); 3.39 (s, 3H).
4.5.1. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]adenine, sodium salt (ODE-(S)-MPMPA) (15)
Deprotection of 10 (procedure D) gave 15 in 73% yield as a
white powder. 1H NMR (CDCl3/methanol-d4) d 8.35 (s, 1H, H-8);
8.22 (s, 1H, H-2); 4.53 (dd, H, H-10a, J1 a2 = 3.3 Hz, Jgem 14.3 Hz);
0
0
4.37 (dd, 1H, H-10b, J1 b2 = 6.6 Hz, Jgem 14.7 Hz); 4.01–3.98 (m,
3H, P–O–CH2– + H-20); 3.87 (dd, 1H, –CHa–P–, JP,CHa = 9.2 Hz,
Jgem = 13.2); 3.70 (dd, 1H, –CHb–P–, JP,CHb = 9.0 Hz, Jgem = 13.0 Hz);
3.57 (t, 2H, –CH2–O–); 3.44 (t, 2H, –O–CH2–); 1.53 (m, 2H, –O–
CH2CH2(CH2)15–); 1.26 (m, 30H, –(CH2)15CH3); 0.89 (t, 3H, –CH3,
J = 7 Hz). MS (ESI+): 614.41 [M+H]+; HRMS (ESIꢁ) calcd for
0
0
4.6.2. (R)-9-[(3-Methoxy-2-hydroxy)propyl]-2,6-diaminopurine
(21)
Compound 21 was synthesized from 2,6-diaminopurine and
(R)-methyl glycidyl ether (procedure A). 41% yield. 1H NMR
(CDCl3/methanol-d4), d 7.73 (s, 1H, H-8); 4.22 (d, 1H, H-10a,
Jgem = 12.4 Hz), 4.06–4.02 (m, 2H, H-10b + H-20); 3.39 (d, 2H, H-30,
J = 3.2 Hz); 3.36 (s, 3H, –OCH3).
C
30H55N5O6P [MꢁH]ꢁ 612.3895, found 612.3897 (E = 0.3 ppm).
HPLC analysis: retention time 22.35 min., purity 96.12%.
4.5.2. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]adenine, sodium salt (ODE-(R)-MPMPA) (16)
Deprotection of 11 gave 16 in 72% yield. 1H NMR (CDCl3/meth-
anol-d4), d 8.24 (s, 1H); 8.21 (s, 1H); 4.43–4.54 (s, 1H); 4.25–4.35
(m, 1H); 3.88–3.98 (m, 3H); 3.80–3.88 (m, 1H); 3.50–3.60 (m,
4H); 3.38–3.48 (m, 3H); 3.37 (s, 3H); 1.49–1.56 (m, 2H); 1.20–
1.35 (m, 30H); 0.88 (t, J=7 Hz, 3H). MS (ESI+): 614.55 [M+H]+,
636.46 [M+Na]+. HRMS (ESIꢁ) calcd for C30H55N5O6P [MꢁH]ꢁ
612.3895, found 612.3900 (E = 0.8 ppm). HPLC analysis: retention
time 21.82 min., purity 95.24%.
4.6.3. (R,S)-9-[(3-Ethoxy-2-hydroxy)propyl]-2,6-diaminopurine
(22)
Compound 22 was synthesized from 2,6-diaminopurine and
(R,S)-ethyl glycidyl ether (procedure A). 71% yield. 1H NMR
(CDCl3/methanol-d4)
d
7.77 (s, 1H, H-8); 4.22 (dd, 1H,
J10a,20 = 3.6 Hz, Jgem = 12.4 Hz, H-10a); 4.06–4.02 (m, 2H, H-10b +
H-20); 3.88 (q, 2H, –OCH2CH3); 3.39 (d, 2H, H-30, J = 3.2 Hz); 1.16
(t, 3H, –OCH2CH3).
4.6.4. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]2,6-diaminopurine, sodium salt (ODE-(S)-
MPMPDAP) (23)
4.5.3. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphono-
methoxy)propyl]adenine, sodium salt (HDP-(S)-MPMPA) (17)
Deprotection of 12 gave 17 in 77% yield. 1H NMR (CDCl3/meth-
anol-d4), d 8.28 (s, 1H); 8.23 (s, 1H); 4.48–4.61 (s, 2H); 4.32–4.37
(m, 2H); 3.91–3.96 (m, 2H); 3.80–3.86 (m, 1H); 3.58–3.64
(m,1H); 3.41–3.57 (m, 3H); 3.30–3.41 (m, 2H); 3.38 (s, 3H);
1.82–1.90 (m, 2H); 1.49–1.55 (m, 2H); 1.18–1.38 (m, 26H); 0.89
(t, J=7 Hz, 3H). MS (ESIꢁ): 598.29 [MꢁH]ꢁ. HRMS (ESIꢁ) calcd for
Compound 23 was synthesized (procedure C) from (S)-9-[(3-
methoxy-2- hydroxy)propyl]2,6-diaminopurine and octadecyloxy-
ethyl p-toluenesulfonyloxymethylphosphonate. 50% yield. 1H NMR
(CDCl3/methanol-d4), d 7.72 (s, 1H); 4.00–4.20 (m, 5H); 3.80–3.90
(m, 1H); 3.58–3.65 (m, 2H); 3.50–3.58 (m, 2H); 3.40–3.50 (m,
1H); 3.44 (s, 3H); 3.30–3.38 (m, 2H); 1.50–1.60 (m, 2H); 1.18–
1.38 (m, 30H); 0.88 (t, J=7 Hz, 3H). MS (ESI): 627.48 [MꢁH]ꢁ,
629.47 [M+H]+. HRMS (ESIꢁ) calcd for C30H56N6O6P [MꢁH]ꢁ
627.4004, found 627.4007 (E = 0.5 ppm). HPLC analysis: retention
time 23.67 min., purity 91.9%.
C
29H53N5O6P [MꢁH]ꢁ 598.3739, found 598.3737 (E = ꢁ0.3 ppm).
HPLC analysis: retention time 22.43 min., purity 93.0%.
4.5.4. Hexadecyloxyethyl (R,S)-9-[(3-ethoxy-2-phosphono-
methoxy)propyl]adenine, sodium salt (HDP-(R,S)-EPMPA) (18)
Deprotection of 13 gave 18 in 92% yield. 1H NMR (CDCl3/metha-
nol-d4), d 8.28 (s, 1H); 8.21 (s, 1H); 4.48–4.53 (s, 1H); 4.34–4.39 (m,
1H); 3.90–4.00 (m, 3H); 3.80–3.86 (m, 1H); 3.58–3.64 (m,1H);
3.44–3.58 (m, 6H); 3.35–3.41 (m, 2H); 1.82–1.90 (m, 2H); 1.49–
1.58 (m, 2H); 1.22–1.38 (m, 26H); 1.20 (t, J=7 Hz, 3H); 0.89 (t,
J=7 Hz, 3H). MS (ESI): 612.44 (MꢁH)ꢁ. HRMS (ESIꢁ) calcd for
4.6.5. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]2,6-diaminopurine, sodium salt (ODE-(R)-
MPMPDAP) (24)
Compound 24 was synthesized (procedure C)from (R)-9-[(3-
methoxy-2- hydroxy)propyl]2,6-diaminopurine and octadecyloxy-
ethyl p-toluenesulfonyloxymethylphosphonate with 49% yield. 1H
NMR (CDCl3/methanol-d4), d 7.75 (s, 1H);4.42–4.51 (m, 1H);
4.00–4.20 (m, 4H); 3.80–3.90 (m, 1H); 3.60–3.65 (m, 2H); 3.50–
3.58 (m, 2H); 3.40–3.50 (m, 1H) 3.49 (s, 3H); 3.30–3.38 (m, 2H);
1.50–1.62 (m, 2H); 1.20–1.38 (m, 30H); 0.88 (t, J=7 Hz, 3H). MS
(ESI+): 629.55 [M+H]+. HRMS (ESIꢁ) calcd for C30H56N6O6P [MꢁH]ꢁ
627.4004, found 627.4007 (E = 0.5 ppm). HPLC analysis: retention
time 23.67, purity 98.4%.
C
30H55N5O6P [MꢁH]ꢁ 612.3895, found 612.3898 (E = 0.5 ppm).
HPLC analysis: retention time 22.60 min., purity 91.8%
4.5.5. Hexadecyloxyethyl (S)-9-[(3-isopropoxy-2-phosphono-
methoxy)propyl]adenine, sodium salt (HDP-(R,S)-IPPMPA) (19)
Deprotection of 14 gave 19 in 75% yield. 1H NMR (methanol-d4)
d
8.35 (s,1H, H-8), 8.25 (s, 1H, H-2), 4.53, (dd, 1H, H-10a,
J1 a2 = 3.4 Hz, Jgem = 14.6 Hz), 4.39 (dd, 1H, H-10b, J1 b2 = 6.6 Hz,
Jgem = 14.6 Hz), 3.93 (t, 2H, P–O–CHa, J = 6.8 Hz), 3.91 (t, 1H, P–O–
CHb, J = 6.4 Hz); 3.85 (dd, –CHa–P–, JP,CHa = 9.0 Hz, Jgem = 13 Hz),
3.66 (dd, 1H, –CHb–P–, JP,Hb = 9.6 Hz, Jgem = 13.2 Hz); 3.59–3.48
(m, 3H, H-30 + H-20), 3.46 (t, 2H, –CH2–O–CH2), 3.37 (t, 2H, –CH2–
O–CH2–), 1.80 (pentet, 2H, –O–CH2–CH2–CH2–O–), 1.51 (m, 2H,
–O–CH2–CH2–(CH2)13–), 1.27 (m, 26H, –(CH2)13–), 1.13 (d, 6H, –
CH(CH3)2), 0.89 (t, 3H, –CH3); MS (ESIꢁ): 626.69 [MꢁH]ꢁ. HRMS
(ESIꢁ) calcd for C31H57N5O6P [MꢁH]ꢁ 626.4052, found 626.4053
0
0
0
0
4.6.6. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphono-
methoxy)propyl]-2,6-diaminopurine, sodium salt (HDP-(S)-
MPMPDAP) (25)
Compound 25 was synthesized (procedure C) from (S)-9-[(3-
methoxy-2- hydroxy)propyl]2,6-diaminopurine and hexadecyl-
oxypropyl p-toluenesulfonyloxymethylphosphonate with 30%
yield. 1H NMR (CDCl3/methanol-d4), d 7.72 (s, 1H); 4.02–4.20 (m,
3H); 3.95–4.02 (m, 2H); 3.78–3.85 (m, 2H); 3.45–3.60 (m, 3H);
3.38–3.45 (m,6H); 1.82–1.95 (m, 2H); 1.45–1.60 (m, 2H);

N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
4623
1.20–1.38 (m, 26H); 0.88 (t, J=7 Hz, 3H). MS (ESI+): 615.50 [M+H]+,
4.7.6. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphono-
methoxy)propyl] -6-O-benzylguanine (32)
637.45 [M+Na]+. HRMS (ESIꢁ) calcd for C29H54N6O6P [MꢁH]ꢁ
613.3848, found 613.3854 (E = 1.0 ppm). HPLC analysis: retention
time 22.98 min., purity 90.3%.
Compound 32 was synthesized (procedure C) from (S)-9-[(3-
methoxy-2-hydroxy)propyl]-6-O-benzylguanine and hexadecyl-
oxypropyl p-toluenesulfonyloxymethylphosphonate. 71% yield.
1H NMR (CDCl3/methanol-d4), d: 7.94 (s, 1H); 7.49–7.55 (m, 2H);
7.24–7.40 (m, 3H); 5.55 (s, 2H);4.30–4.40 (m, 1H); 4.17–4.22 (m,
1H); 3.80–3.92 (m, 3H); 3.72–3.92 (m, 1H); 3.55–3.62 (m, 1H);
3.40–3.52 (m, 4H); 3.28–3.40 (m, 2H); 3.37 (s, 3H); 1.75–1.85 (m,
2H); 1.44–1.60 (m, 2H); 1.16–1.38 (m, 26H); 0.89 (t, J=7 Hz, 3H).
MS (ESI): 706.50 [M+H]+.
4.6.7. Hexadecyloxypropyl (R,S)-9-[3-ethoxy-2-phosphono-
methoxy)propyl-2,6-diaminopurine, sodium salt (HDP-(R,S)-
EPMPDAP) (26)
Compound 26 was synthesized (procedure C) from (R,S)-9-[(3-
ethoxy-2- hydroxy)propyl]2,6-diaminopurine and hexadecyloxy-
propyl p-toluenesulfonyloxymethylphosphonate with 22% yield.
1H NMR (CDCl3/methanol-d4), d 7.40 (s, 1H); 4.44–4.52 (m, 1H);
4.18–2.28(m, 1H); 3.91–4.10 (m, 3H); 3.80–3.90 (m, 1H); 3.45–
3.60 (m, 5H); 3.38–3.45 (m, 4H); 1.82–1.95 (m, 2H); 1.45–1.60
(m, 2H); 1.15–1.38 (m, 28H); 0.88 (t, J=7 Hz, 3H). MS (ESIꢁ):
627.53 [MꢁH]ꢁ. HRMS (ESIꢁ) calcd for C30H56N6O6P [MꢁH]ꢁ
627.4004, found 627.4008 (E = 0.6 ppm). HPLC analysis: retention
time 23.37 min., purity 96.4%.
4.7.7. Hexadecyloxypropyl (R,S)-9-[3-ethoxy-2-phosphono-
methoxy)propyl]-6-O-benzylguanine (33)
Compound 33 was synthesized (procedure C) from (R,S)-9-[(3-
methoxy-2-hydroxy)propyl]-6-O-benzylguanine and hexadecyl-
oxypropyl p-toluenesulfonyloxymethylphosphonate. 42% yield).
1H NMR (CDCl3/methanol-d4), d 7.95 (s, 1H); 7.48–7.52 (m, 2H);
7.30–7.40 (m, 3H); 5.56 (s, 2H);4.34–4.40 (m, 1H); 4.19–4.26 (m,
1H); 3.77–3.93 (m, 4H); 3.58–3.66 (m, 1H); 3.47–3.55 (m, 5H);
3.35–3.45 (m, 3H); 1.78–1.85 (m, 2H); 1.48–1.55 (m, 2H); 1.17–
1.28 (m, 29H); 0.89 (t, J = 7 Hz, 3H). MS (ESI): 718.46 [MꢁH]ꢁ.
4.7. Guanine derivatives
4.7.1. (S)-9-[(3-Methoxy-2-hydroxy)propyl]-6-O-benzylguanine
(27)
Compound 27 was synthesized from 6-O-benzylguanine (APAC
Pharmaceutical LLC, Columbia, MD) and (S)-methyl glycidyl ether
(procedure A). 49% yield. 1H NMR (CDCl3/methanol-d4), d 7.75 (s,
1H); 7.49–7.51 (m, 2H); 7.29–7.40 (m, 3H); 5.55 (s, 2H); 4.23–
4.32 (m, 1H); 4.05–4.14 (m, 2H); 3.39–3.41 (m, 2H); 3.39 (s, 3H).
4.8. General procedure E. Synthesis of alkoxyalkyl-9-[(3-alkoxy-
2-phosphonomethoxy)propyl]guanine (34–37)
The protected guanine compounds (30–33) (0.71 mmol) were
added to 10% trifluoroacetic acid/CH2Cl2 and the mixture was stir-
red at room temperature for 2 days. The solvent was removed in
vacuo and the residue purified by flash column chromatography
on silica gel. The column was eluted with 20% MeOH/CH2Cl2 and
the crude products were recrystallized from water to give the al-
4.7.2. (R)-9-[(3-Methoxy-2-hydroxy)propyl] -6-O-benzylguanine
(28)
Compound 28 was synthesized from 6-O-benzylguanine and
(R)-methyl glycidyl ether (procedure A). 44% yield. 1H NMR
(CDCl3/methanol-d4), d 7.74 (s, 1H); 7.47–7.51 (m, 2H); 7.29–7.40
(m, 3H); 5.55 (s, 2H); 4.40–4.60 (m, 1H); 4.05–4.14 (m, 2H);
3.32–3.45 (m, 2H); 3.39 (s, 3H).
koxyalkyl
9-[(3-alkoxy-2-phosphonomethoxy)propyl]guanine
derivatives (34–37).
4.8.1. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]guanine (ODE-(S)-MPMPG) (34)
4.7.3. (R,S)-9-[(3-Ethoxy-2-hydroxy)propyl]-6-O-benzylguanine
(29)
Deprotection of 30 gave 34 as a white powder. 93% yield. 1H
NMR (CDCl3/methanol-d4), 7.82 (s, 1H); 4.24–4.36 (m, 1H); 4.10–
4.28 (m, 1H); 3.95–4.05 (m, 2H); 3.78–3.90 (m, 2H); 3.62–3.73
(m, 1H); 3.52–3.60 (m, 2H); 3.40–3.50 (m, 2H); 3.25–3.40 (m,
3H); 1.45–1.60 (m, 2H); 1.18–1.38 (m, 30H); 0.89 (t, J = 7 Hz, 3H).
MS (ESI): 628.44 [MꢁH]ꢁ. HRMS (ESIꢁ) calcd for C30H55N5O7P
[MꢁH]ꢁ 628.3845, found 628.3846 (E = 0.2 ppm). HPLC analysis:
retention time 21.10, purity 96.2%.
Compound 29 was synthesized from 6-O-benzylguanine and
(R,S) ethyl glycidyl ether (procedure A). 51% yield. 1H NMR
(CDCl3/methanol-d4), d 7.76 (s, 1H); 7.50–7.52 (m, 2H); 7.32–7.40
(m, 3H); 5.52 (s, 2H); 4.17–4.21 (m, 1H); 3.95–4.00 (m, 2H);
3.44–3.50 (m, 2H); 3.34–3.38 (m, 2H); 1.16 (t, J=7 Hz, 3H).
4.7.4. Octadecyloxyethyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]-6-O-benzylguanine (30)
4.8.2. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]guanine (ODE-(R)-MPMPG) (35)
Compound 30 was synthesized (procedure C) from (S)-9-[(3-
methoxy-2-hydroxy)propyl]-6-O-benzylguanine and octadecyl-
oxyethyl p-toluenesulfonyloxymethylphosphonate. 26% yield. 1H
NMR (CDCl3/methanol-d4), d 7.93 (s, 1H); 7.50–7.56 (m, 2H);
7.31–7.40 (m, 3H); 5.55 (s, 2H); 4.24–4.36 (m, 1H); 3.93–4.22 (m,
1H); 3.75–3.98 (m, 4H); 3.60–3.70 (m, 4H); 3.30–3.60 (m, 8H);
1.42–1.60 (m, 2H); 1.18–1.38 (m, 30H); 0.89 (t, J=7 Hz, 3H). MS
(ESI): 720.51 [M+H]+.
Deprotection of 31 gave 35 as a white powder. 67% yield. 1H
NMR (CDCl3/methanol-d4), 8.07 (s, 1H, H-8); 7.51 (s, 2H, –NH2);
4.34 (dd, 1H, H-10a, J1 a2 = 3.9 Hz, Jgem = 14.6 Hz); 4.13 (dd, 1H,
0
0
H-10b, J1 b2 = 6.4 Hz, Jgem = 14.3 Hz); 4.00 (m, 2H, –P–O–CH2–);
3.87 (dd, 1H, –CHa–P–, JP,CHa = 8.7 Hz, Jgem = 12.9 Hz); 3.68 (dd,
1H, –CHb–P–, JP,CHb = 9.6 Hz, Jgem = 12.8 Hz); 3.59 (t, 2H, –CH2–O–
CH2); 3.46 (d + t, 4H, –CH2–O–CH2 + H-30); 3.38 (s, 3H, –OCH3);
1.50–1.62 (m, 2H, –O–CH2CH2(CH2)15–); 1.27 (m, 30H, –(CH2)15–);
0.89 (t, J = 7 Hz, 3H, –CH3). MS (ESI+): 630.29 [M+H]+; HRMS (ESIꢁ)
0
0
4.7.5. Octadecyloxyethyl (R)-9-[(3-methoxy-2-phosphono-
methoxy)propyl] -6-O-benzylguanine (31)
calcd for
C
30H55N5O7P [MꢁH]ꢁ 628.3845, found 628.3843
Compound 31 was synthesized (procedure C) from (R)-9-[(3-
methoxy-2-hydroxy)propyl]- 6-O-benzylguanine and octadecyl-
oxyethyl p-toluenesulfonyloxymethylphosphonate. 83% yield. 1H
NMR (CDCl3/methanol-d4), d 7.93 (s, 1H); 7.52–7.47 (m, 2H);
7.23–7.38 (m, 3H); 5.55 (s, 2H); 4.18–4.38 (m, 2H); 3.75–3.98 (m,
4H); 3.55–3.65 (m, 1H); 3.43–3.50 (m, 3H); 3.30–3.43 (m, 8H);
1.45–1.60 (m, 2H); 1.18–1.38 (m, 30H); 0.89 (t, J=7 Hz, 3H). MS
(ESI): 718.54 [MꢁH]ꢁ.
(E = ꢁ0.3 ppm). HPLC analysis: retention time 22.33 min., purity
92.9%.
4.8.3. Hexadecyloxypropyl (S)-9-[3-methoxy-2-phosphono-
methoxy)propyl]guanine (HDP-(S)-MPMPG) (36)
Deprotection of 32 gave 36 as a white powder. 93% yield. 1H
NMR (CDCl3/methanol-d4), 7.83 (s, 1H); 4.26–4.31 (m, 1H); 4.06–
4.11 (m, 1H); 3.93–3.98 (m, 2H); 3.81–3.86 (m, 2H); 3.60–3.63

4624
N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
(m, 2H); 3.48–3.57 (m, 3H); 3.35–3.44 (m, 2H); 3.37 (s, 3H); 1.84–
1.89 (m, 2H); 1.52–1.58 (m, 2H); 1.15–1.40 (m, 26H); 0.88 (t,
J=7 Hz, 3H). MS (ESI): 616.45 [M+H]+, 638.40 [M+Na]+. HRMS (ESIꢁ)
4.10.2. (S)-9-[(3-Methoxy-2-hydroxy)propyl]-6-O-
methylguanine (42)
Reaction of 6-O-methylguanine (Aldrich Chem.) with (S)-meth-
ylglycidyl ether (Procedure A) gave compound 42. 78% yield. 1H
NMR (methanol-d4) d 7.81 (s, 1H, H-8); 4.35 (dd, 2H, H-10); 4.15
(m, 1H, H-20); 4.05 (s, 3H, Ar-OCH3); 3.39 (d, 2H, H-30); 3.35 (s,
3H, –OCH3). MS (ESI): m/z 254.08 [M+H]+.
calcd for
C
29H53N5O7P [MꢁH]ꢁ 614.3688, found 614.3687
(E = ꢁ0.2 ppm). HPLC analysis: retention time 20.55 min., purity
93.6%.
4.8.4. Hexadecyloxypropyl (R,S)-9-[3-ethoxy-2-phosphono-
methoxy)propyl]guanine (HDP-(R,S)-EPMPG) (37)
4.10.3. Hexadecyloxypropyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]-6-methoxypurine (HDP-(S)-MPMPMP) (43)
Reaction of compound 41 with hexadecyloxypropyl p-toluene-
sulfonyloxymethylphosphonate according to procedure C afforded
compound 43. 29% yield. 1H NMR (methanol-d4) d 8.51 (s, 1H,
Deprotection of 33 gave 37. 88% yield. 1H NMR (CDCl3/metha-
nol-d4), 7.85 (s, 1H); 4.28–4.33 (m, 1H); 4.11–4.17 (m, 1H); 3.92–
3.97 (m, 2H); 3.79–3.86 (m, 2H); 3.63–3.92 (m, 1H); 3.47–3.56
(m, 5H); 3.34–3.42 (m, 3H); 1.83–1.90 (m, 2H); 1.52–1.57 (m,
2H); 1.22–1.40 (m, 26H); 1.20 (t, J=7 Hz, 3H); 0.88 (t, J=7 Hz, 3H).
MS (EI): 628.40 [MꢁH]ꢁ. HRMS (ESIꢁ) calcd for C30H55N5O7P
[MꢁH]ꢁ 628.3845, found 628.3848 (E = 0.5 ppm). HPLC analysis:
retention time 20.92 min., purity 95.1%.
H-8); 8.44 (s, 1H, H-2); 4.56 (dd, 1H, H-10a , J1 a,2 = 3.6 Hz,
0
0
Jgem = 14.0 Hz); 4.44 (dd, 1H, H-10b, J1 b,2 = 6.6 Hz, Jgem = 14.6 Hz);
4.17 (s, 3H, Ar–OCH3); 3.96 (m, 1H, H-20); 3.86 (t, 1H, P–O–CHa,
J = 6.4 Hz); 3.84 (t, 1H, P–O–CHb, J = 6.4 Hz); 3.78 (dd, 1H, –CHa–
P–, JP,CH = 9.2 Hz, Jgem = 12.8 Hz); 3.60 (dd, 1H, –CHb–P–,
JP,CH = 9.6 Hz, Jgem = 12.8 Hz); 3.44 (m, 1H, H-20); 3.43 (t, 2H, –
CH2–O–CH2–); 3.36 (t, 2H, –CH2–O–CH2–); 3.32 (s, 3H, –OCH3);
1.76 (pentet, 2H, –O–CH2CH2CH2–O–); 1.49 (m, 2H, –CH2–O–
CH2CH2(CH2)13–); 1.28 (m, 26H, –(CH2)13–); 0.89 (t, 3H, –CH3).
MS (ESIꢁ) m/z 613.46 [MꢁH]ꢁ. HRMS (ESIꢁ) calcd for C30H54N4O7P
[MꢁH]ꢁ 613.3736, found 613.3739 (E = 0.5 ppm).
0
0
4.9. Cytosines
4.9.1. (S)-1-[(3-Methoxy-2-hydroxy)propyl]-N4-monomethoxy-
tritylcytosine (38)
Compound 38 was synthesized from N4-monomethoxytritylcy-
tosine4 and (S)-methyl glycidyl ether (procedure A). 91% yield. 1H
NMR (CDCl3/methanol-d4), d 7.45–7.67 (m, 14H); 7.17 (d, J=6 Hz,
1H); 5.82 (d, J=6 Hz, 1H); 4.30–4.40 (m, 2H); 4.12 (s, 3H); 3.80–
3.92 (m, 1H); 3.65–3.75 (m, 2H); 3.65 (s, 3H).
4.10.4. Hexadecyloxypropyl (S)-9-[(3-methoxy-2-phosphono-
methoxy)propyl]-6-O-methylguanine (HDP-(S)-MPMPOMG)
(44)
Reaction of compound 42 with hexadecyloxypropyl p-toluene-
sulfonyloxymethylphosphonate according to procedure C afforded
compound 44. 34% yield. 1H NMR (methanol-d4) d 7.95 (s, 1H, H-8),
4.9.2. Octadecyloxyethyl (S)-1-[(3-methoxy-2-
phosphonomethoxy)propyl]-N4-monomethoxytritylcytosine
(39)
4.34 (dd, 1H, H-10a, J1 a2 = 4 Hz, Jgem = 14.4 Hz); 4.22 (dd, 1H, H-10b,
0
0
Compound 39 was synthesized from (S)-9-[(3-methoxy-2-
hydroxy)propyl]-N4-monomethoxytritylcytosine (38) and octade-
cyloxyethyl p-toluenesulfonyloxymethylphosphonate (procedure
C). 45% yield 1H NMR (CDCl3/methanol-d4), d 7.10–7.40 (m, 14H);
6.85 (d, J=6 Hz, 1H); 5.52 (d, J=6 Hz, 1H); 4.20–4.39 (m, 2H);
3.77–4.02 (m, 4H); 3.55–3.65 (m, 1H); 3.43–3.52 (m, 3H); 3.30–
3.45 (m, 8H); 1.45–1.65 (m, 2H); 1.18–1.40 (m, 30H); 0.89 (t,
J=7 Hz, 3H). MS (ESI): 860.55 [MꢁH]ꢁ.
0
0
J1 b2 = 6.4 Hz, Jgem = 14.4 Hz); 4.04 (s, 3H, Ar–OCH3); 3.91 (m, 1H,
H-20); 3.87 (t, 1H, P–O–CHa–, J = 6.4 Hz); 3.86 (t, 1H, P–O–CHb–,
J = 6.4 Hz); 3.75 (dd, 1H, –CHa–P–, JCH,P = 9.4 Hz, Jgem = 13 Hz);
3.64 (dd, 1H, –CHb–P–, JCH,P = 9.2 Hz, Jgem = 12.8 Hz); 3.44 (t, 2H,
–CH2–O–); 3.43 (d, 2H, H-30); 3.35 (t, 2H, –O–CH2); 3.34 (s, 3H, –
OCH3); 1.78 (pentet, 2H, –O–CH2CH2CH2–O–); 1.50 (m, 2H, –
OCH2CH2(CH2)13–); 1.27 (m, 26H, (CH2)13); 0.89 (t, 3H, –CH3). MS
(ESI): m/z 630.35 [M+H]+; 628.37 [MꢁH]ꢁ. HRMS (ESIꢁ) calcd for
C30H55N5O7P [MꢁH]ꢁ 628.3845, found 628.3847 (E = 0.3 ppm).
HPLC analysis: retention time 21.60 min., purity 97.0%.
4.9.3. Octadecyloxyethyl (S)-1-[(3-methoxy-2-phosphono-
methoxy)propyl]cytosine, sodium salt (40) (ODE-(S)-MPMPC)
Octadecyloxyethyl (S)-1-[(3-methoxy-2-phosphonomethoxy)-
propyl]-N4-monomethoxytritylcytosine (39) (0. 26 g, 0.60 mmol)
was added to 80% aq acetic acid and heated to 60 °C for 2 h. After
cooling, the solvent was removed in vacuo and the residue purified
by flash column chromatography on silica gel. Elution with 20%
MeOH/CH2Cl2 gave the product (0.1 g, 55%). 1H NMR (CDCl3/meth-
anol-d4) d 7.80 (d, J = 6 Hz, 1H); 6.00 (d, J = 6 Hz, 1H); 4.04–4.15 (m,
4H); 3.55–3.68 (m, 3H); 3.42–3.53 (m, 2H); 3.35–3.42 (m, 4H);
1.50–1.65 (m, 2H); 1.18–1.38 (m, 30H); 0.88 (t, J = 7 Hz, 3H). MS
(ESI+): 590.33 [M+H]+, 612.34 [M+Na]+. HRMS (ESIꢁ) calcd for
4.11. Assays
4.11.1. HCV antiviral activity
HCV assays were carried out as previously described using
10,000 FEO replicon cells (BM4–5 or JFH-1 based) per well in 96-
well plates.14 Cells and compounds were incubated for 48–72 h
with all conditions run in triplicate. Additionally, three to four
experimental replicates were completed per compound. Luciferase
activity was determined using a micro-plate luminometer (Veritas
Microplate Luminometer, Turner Biosystems) according to the
manufacturer’s instructions (Brightglo, Promega). Relative light
units (RLU) for each condition were used to generate a dose re-
sponse for each compound using Prism (version 4, GraphPad soft-
ware). The cytotoxicity of each compound was determined using a
fluorescent cell viability and death assay (MultiTox-Fluor, Prome-
C
29H55N3O7P [MꢁH]ꢁ 588.3783, found 588.3784 (E = ꢁ0.2 ppm).
HPLC analysis: retention time 22.75 min., purity 90.9%.
4.10. 6-Methoxypurine derivatives
4.10.1. (S)-9-[(3-Methoxy-2-hydroxy)propyl]-6-methoxypurine
(41)
ga). All compounds were tested at concentrations up to 100
(0.3% DMSO final concentration)
lM
Reaction of 6-methoxypurine (TCI America, Portland, OR) with
(S)-methyl glycidyl ether according to procedure A gave compound
41. 67% yield. 1H NMR (methanol-d4) d 8.51 (s, 1H, H-8), 8.24 (s, 1H,
4.11.2. HIV-1 inhibition assays
MT-2 cells (AIDS Research and Reference Reagent Program,
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health) were maintained in RPMI 1640 supplemented with
10% FBS (JRH Biosciences, Lenexa, Kans.), 10 mM HEPES buffer,
0
0
H-2), 4.74 (dd, 1H, J1 a2 = 3.8 Hz, Jgem=14.2 Hz), 4.28 (dd, 1H,
0
0
J1 b2 = 8 Hz, Jgem = 14.2 Hz), 4.18 (s, 3H, Ar–OCH3), 4.14 (m, 1H,
H-20), 3.42 (d, 2H, J3 2 = 5.2 Hz), 3.37 (s, 3H, –CH2–OCH3).
0
0

N. Valiaeva et al. / Bioorg. Med. Chem. 19 (2011) 4616–4625
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4625
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threefold serial drug dilutions (three wells per dilution). Four days
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a commercial enzyme-linked immunosorbent assay (ELISA) (Per-
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compound is expressed as the EC50, which is the concentration
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toxicity, MT-2 cells were incubated with drug for 72 hrs and har-
vested. Flow count beads (Beckman Coulter, Miami, FL) were
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and analysis using an Epics Elite flow cytometer (Beckman Coul-
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Acknowledgements
This work was supported in part by NIH Grants AI-076558
(R.T.S.), AI-074057, AI-071803, AI-069989 (K.Y.H.) and contract
N01-AI-30049 (M.N.P.). The authors wish to thank Krysten Jones,
Kathy A. Aldern, Julissa Trahan, Kathy A. Keith and Caroll B. Hart-
line for technical assistance.
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