Synthesis and anti-HIV evaluation of new acyclic phosphonate nucleotide analogues and their bis(SATE) derivatives

Article abstract of DOI:10.1080/15257770.2010.495958

This article describes a very simple route for synthesizing novel lipophilic phosphonate bis(t-bu-SATE) prodrugs of acyclic cyclopentenylated nucleosides such as adenine 17 and cytosine 18. The key intermediate 6 was constructed via a ring-closing metathesis of compound 5, which could be readily prepared from diethylmalonate 4. The chemical stability of the bis(SATE) derivatives was tested at neutral (pH = 7.2) and slightly acid (milli-Q water, pH = 5.5) pH. The synthesized compounds were evaluated as potential antiviral agents against HIV-1 virus.

Full text of DOI:10.1080/15257770.2010.495958

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Synthesis and Anti-HIV Evaluation of
New Acyclic Phosphonate Nucleotide
Analogues and Their Bis(SATE)
Derivatives
a
a
Hua Li & Joon Hee Hong
a
BK-21 Project Team, College of Pharmacy , Chosun University ,
Kwangju, Republic of Korea
Published online: 19 Jul 2010.
To cite this article: Hua Li & Joon Hee Hong (2010) Synthesis and Anti-HIV Evaluation of New Acyclic
Phosphonate Nucleotide Analogues and Their Bis(SATE) Derivatives, Nucleosides, Nucleotides and
Nucleic Acids, 29:8, 581-590, DOI: 10.1080/15257770.2010.495958
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Nucleosides, Nucleotides and Nucleic Acids, 29:581–590, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.495958
SYNTHESIS AND ANTI-HIV EVALUATION OF NEW ACYCLIC
PHOSPHONATE NUCLEOTIDE ANALOGUES AND THEIR
BIS(SATE) DERIVATIVES
Hua Li and Joon Hee Hong
BK-21 Project Team, College of Pharmacy, Chosun University, Kwangju, Republic of Korea
2
This article describes a very simple route for synthesizing novel lipophilic phosphonate bis(t-bu-
SATE) prodrugs of acyclic cyclopentenylated nucleosides such as adenine 17 and cytosine 18. The
key intermediate 6 was constructed via a ring-closing metathesis of compound 5, which could be
readily prepared from diethylmalonate 4. The chemical stability of the bis(SATE) derivatives was
tested at neutral (pH = 7.2) and slightly acid (milli-Q water, pH = 5.5) pH. The synthesized
compounds were evaluated as potential antiviral agents against HIV-1 virus.
Keywords Antiviral agent; SATE prodrug; phosphonate nucleoside
INTRODUCTION
Emerging drug-resistant virus strains and toxicity are major problems
with antiviral chemotherapy. A number of structurally modified nucleosides
have been synthesized in an attempt to overcome these drawbacks. Fun-
damental modifications of the classical pentofuranose moiety have been
designed, and many sugar modified nucleoside analogues have shown excel-
lent antiviral or antitumor activity. Although structure-activity relationship
studies have not led to a pharmacophore model for the antiviral activities
of nucleosides, some structural features have been particularly successful.
[1]
Recently, a number of acyclic nucleoside, analogues with phosphonic acid
groups have been synthesized and evaluated for antiviral activity. Among
them, PMEA 1,[ PMPA 2, and HPMPC 3 exhibit potent antiviral ac-
tivity against HIV, HBV, and HSV (Figure 1). Unlike nucleoside agents, a
phosphonate nucleoside has the advantage of skipping the requisite initial
2]
[3]
[4]
[5]
phosphorylation, which is a crucial step for the activation of nucleosides.
However, the poor oral bioavailability of these nucleoside analogues are
Received 6 April 2010; accepted 21 December 2010.
Address correspondence to Joon Hee Hong, College of Pharmacy, Chosun University, Kwangju
501-759, Republic of Korea. E-mail: hongjh@chosun.ac.kr
5
81

5
82
H. Li and J. H. Hong
NH2
N
NH2
N
N
N
N
N
N
O
N
O
HO P
O
HO P
O
OH
OH
CH3
PMEA (1)
PMPA (2)
NH2
N
N
O
O
O
HO P
OH
OH
HPMPC (3)
FIGURE 1 Rationale to the design of the target nucleotide analogues.
due to the phosphonate negative charges present in nucleoside phosphonic
acid at physiological pH. Therefore, the concept of temporarily masking
these charges with neutral groups to form more lipophilic derivatives ca-
pable of crossing the gastrointestinal wall and reverting back to the parent
[6]
nucleoside phosphonic acid was attempted. Since the ionic character of
a phosphonic acid would present an obstacle for cellular permeability, an
S-acyl-2-thioethyl (SATE) prodrug was prepared. Esterification of a phospho-
nic acid with two SATE groups is a feasible strategy to deliver a phosphate
[7]
or phosphonate drug into cells. Usually, phosphonic acid nucleosides re-
quire an endocytosis-like process[ or the ATP membrane receptor to
cross the cellular membrane. We therefore applied the bis(SATE) approach
to novel acyclic nucleoside phosphonic acids. Here, we report on the synthe-
sis, antiviral activity, and stability of the t-bu-SATE prodrugs of novel acyclic
nucleoside analogues.
8]
[9]
RESULTS AND DISCUSSION
For the synthesis of carboacyclic nucleosides, the commercially available
diethyl malonate 4 was selected as a starting material. As shown in Scheme 1,
the synthetic route is very simple and straightforward. Double alkylation
of the active methylene group of 4 and ring-closing metathesis (RCM) of
corresponding methyl-allyl 5 provided dimethyl-cyclopentene derivative 6 in

New Acyclic Bis(SATE) Nucleoside Prodrugs
583
O
O
O
O
HO
O
O
ii
OH
i
iii
EtO
EtO
OEt
OEt
EtO
OEt
4
6
7
5
iv
i-PrO
i-PrO
v
vi
OR
i-PrO P
O
HO
OR
i-PrO P
O
OH
O
R = TBDMS
O
9
1
0
8
vii
B
i-PrO
i-PrO
viii
i-PrO P
O
i-PrO P
O
OMs
O
O
11
1
1
2: B = Adenine
3: B = Cytosine
SCHEME 1 Synthesis of phosphonate nucleosides. Reagents: i) NaH, 3-cholor-2-methyl-propene, THF;
ii) Grubb’s catalyst (II), benzene; iii) LiAIH4, THF; iv) TBDMSCI, imidazole, CH2Cl2; v) Diisopropyl
bromomethyphosphonate, LiOt-Bu, Lil, DMF; vi) TBAF, THF; vii) MsCl, TEA, CH2Cl2; viii) adenine,
cytosine K2CO3, 18-Crown-6, DMF.
a high yield.[10] Reduction of the ester functional group of 6, followed by a
monosilylation of diol 7 provided the alcohol derivative 8.
For the synthesis of phosphonate nucleosides, the hydroxyl group of
8
was phosphonated by treating with diisopropyl bromomethylphospho-
[11]
nate
in anhydrous DMF to give the phosphonate intermediate 9 (Scheme
1
), which was readily desilylated by TBAF to provide 10. Compound 10 was ac-
tivated by methanesulfonylation with MsCl and triethylamine in anhydrous
CH Cl to give 11, which was coupled with natural base (adenine, cyto-
2
2
sine) under nucleophilic S 2 substitution conditions (K CO , 18-Crown-6,
N
2
3
[
12]
DMF)
to give the dimethylcyclopentene nucleoside phosphonate 12 and
1
3, respectively. The diisopropyl protecting groups of the phosphonates were
[13]
readily removed using trimethylsilylbromide
to give nucleoside phospho-
nic acids 15 and 16. To synthesize the thioester-protected analogues, phos-
phonic acid nucleosides were reacted with thioester 14 in the presence of
[
14]
1
-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT)
to provide the
final t-bu-SATE prodrugs 17 and 18 (Scheme 2). The newly synthesized phos-
phonic nucleoside analogues 15, 16, 17, and 18 were assayed in the MT-4 cell
line for anti-HIV activity using the previously reported assay system.[ The
15]

5
84
H. Li and J. H. Hong
B
HO
B
i-PrO
i
HO P
O
O
i-PrO P
O
O
1
1
5: B = Adenine
6: B = Cytosine
1
1
2: B = Adenine
3: B = Cytosine
ii
O
OH
S
S
14
B
O
S
O
O
O P
O
O
1
1
7: B = Adenine
8: B = Cytosine
SCHEME 2 Synthesis of SATE prodrug of phosphonic acid nucleoside. Reagents: i) TMSBr, CH3CN; ii)
4, 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole, pyridine.
1
tested prodrugs enhanced the in vitro anti-HIV activity of parent phosphonic
acid as well as the cytotoxicity (Table 1) via an increase in cellular uptake
followed by intracellular release of parent phosphonic acids.
To measure the relative chemical stabilities of the SATE prodrugs, the
percent of decomposition was briefly measured for SATE prodrugs 17, 18
◦
(
i) at 37 C in milli-Q water (pH = 5.5) and (ii) in pH = 7.2 buffer using the
[7]
Gosselin’s method (Table 2).
TABLE 1 Antiviral activity of the synthesized compounds
HIV-1
Cytotoxicity
Compd
EC50 (µM)
CC50 (µM)
15
16
17
18
97
99
41
>100
>100
47
39
44
bis(POM)PMEA
0.09
0.78
Bis(POM)PMEA: bis(pivaloyloxymethyl)9-[2-(phosphonomethoxy)ethyl]
adenine.
EC50: Concentration (µM) required to inhibit the replication of HIV-1 by 50%.
CC50: Concentration (µM) required to reduce the viability of unaffected cells
by 50%.

New Acyclic Bis(SATE) Nucleoside Prodrugs
585
TABLE 2 Percentage of decomposition after 24 hours of
incubation at two different media
Compd
Water
pH = 7.2
1
1
7
8
1.6%
1.5%
3.5%
3.2%
Water condition: Milli-Q, pH = 5.5.
pH = 7.2 condition: Ammonium acetate buffer, 0.02 M.
CONCLUSION
We have synthesized and tested the anti-HIV activity and chemical stabil-
ity of novel SATE prodrugs of cyclopentene phosphonate nucleosides. The
tested prodrugs showed increased anti-HIV activity over parent phosphonic
acids. Also, the synthesized prodrugs were relatively stable in neutral and
weakly acidic media.
EXPERIMENTAL
Melting points were determined on a Mel-temp II laboratory device and
are uncorrected. NMR spectra were recorded on a JEOL 300 Fourier trans-
form spectrometer (JEOL, Tokyo, Japan); chemical shifts are reported in
parts per million (δ) and signals are reported as s (singlet), d (doublet), t
(
triplet), q (quartet), m (multiplet), and dd (doublet of doublets). Ultra-
violet (UV) spectra were obtained on a Beckman DU-7 spectrophotometer
Beckman, South Pasadena, CA, USA). The elemental analyses were per-
(
formed using a Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT,
USA). Thin layer chromatography (TLC) was performed on Uniplates (sil-
ica gel) purchased from Analtech Co. (7558, Newark, DE, USA). All reac-
tions were performed under an atmosphere of nitrogen unless specified. Dry
dichloromethane, benzene, and pyridine were obtained by distillation from
CaH . Dry THF was obtained by distillation from Na and benzophenone
2
immediately prior to use.
2
,2-Bis-(2-methylallyl) malonic acid diethyl ester (5): To a stirred suspen-
sion of sodium hydride (1.87 g, 0.078 mol) in tetrahydrofuran (150 mL)
◦
was slowly added diethyl malonate 4 (5 g, 0.0312 mol) at 0 C and stirred for
2
2
.5 hours at room temperature. To this mixture was slowly added 3-chloro-
-methyl-propene (5.9 g, 65.5 mmol) at 0 C and stirred overnight at room
◦
temperature. The mixture was quenched with saturated NaHCO solution
3
(
10 mL) and further diluted with water (200 mL). The mixture was extracted
using EtOAc (300 mL), and the organic layer was washed with brine, dried
over MgSO , filtered, and then concentrated. The residue was purified by sil-
4
ica gel column chromatography (EtOAc/hexane, 1:15) to give 5 (7.1 g, 85%)

5
86
H. Li and J. H. Hong
1
as a colorless oil: H NMR (CDCl , 300 MHz) δ 4.84 (s, 2H), 4.73 (s,
3
2
7
1
H), 4.17 (q, J = 7.0 Hz, 4H), 2.78 (s, 4H), 1.68 (s, 6H), 1.25 (t, J =
13
.0 Hz, 6H); C NMR (CDCl ) δ 171.5, 141.0, 115.1, 61.2, 56.7, 40.5, 23.6,
3
3.9; Anal. Calc. for C H O : C, 67.14; H, 9.01. Found: C, 67.27; H, 8.94.
15
24
4
3
,4-Dimethylcyclopent-3-ene-1,1-dicarboxylic acid diethyl ester (6): To a
stirred solution of 5 (2.79 g, 10.4 mmol) in anhydrous benzene (10 mL)
◦
was added Grubbs catalyst, 2nd generation (50 mg, 58.89 µmol) at 0 C
and refluxed for 12 hours. The mixture was concentrated under reduced
pressure, and the residue was purified by silica gel column chromatography
1
(
(
(
EtOAc/hexane, 1:12) to give 6 (2.25 g, 90%) as a colorless oil: H NMR
CDCl , 300 MHz) δ 4.19 (q, J = 6.9 Hz, 4H), 2.92 (s, 4H), 1.57 (s, 6H), 1.22
3
13
t, J = 6.9 Hz, 6H); C NMR (CDCl ) δ 172.6, 128.0, 61.3, 45.8, 14.0, 13.3;
3
Anal. Calc. for C H O : C, 64.98; H, 8.39. Found: C, 65.15; H, 8.28.
13
20
4
(
1-Hydroxymethyl-3,4-dimethylcyclopent-3-enyl) methanol (7): To a sus-
pension of lithium aluminum hydride (0.85 g, 22.55 mmol) in dry tetrahydro-
furan (30 mL) was added a solution of diethylester 6 (2.58 g, 10.74 mmol) in
◦
dry tetrahydrofuran (15 mL) drop wise at 0 C. The resulting suspension was
◦
stirred overnight at 0 C. The suspension was quenched with water (0.86 mL),
1
5% sodium hydroxide (0.86 mL), and water (2.58 mL) at the same tem-
perature. The mixture was stirred at room temperature for 2 hours. The
white gel suspension was filtered through a pad of Celite and concentrated
in vacuo. The residue was purified by silica gel column chromatography
1
(
EtoAc/n-Hexane, 4:1) to give diol 7 (1.38 g, 82%) as a colorless oil: H
13
NMR (CDCl , 300 MHz) δ 3.68 (s, 4H), 2.14 (s, 4H), 1.58 (s, 6H); C NMR
3
(
6
CDCl ) δ 128.77, 70.55, 145.41, 44.08, 13.73; Anal. Calc. for C H O : C,
9.19; H, 10.32. Found: C, 69.23; H, 10.29.
3
9
16
2
(
t-Butyldimethylsilanyloxymethyl) 3,4-dimethyl-cyclopent-3-enyl] metha
nol (8): t-Butyldi methylsilyl chloride (1.33 g, 8.83 mmol) was added slowly
to a solution of 7 (1.38 g, 8.83 mmol) and imidazole (1.20 g, 17.67 mmol) in
◦
CH Cl (20 mL) at 0 C, and stirred for 5 hours at the same temperature. The
2
2
solvent was evaporated under reduced pressure. The residue was extracted
twice with EtOAc and water. The combined organic layer was dried over
anhydrous MgSO , filtered, and concentrated under reduced pressure. The
4
residue was purified by silica gel column chromatography (EtOAc/hexane,
1
1
:15) to give 8 (2.27 g, 95%) as a colorless syrup: H NMR (CDCl , 300 MHz)
3
δ s (3.53, 4H), 2.13–1.95 (m, 4H), 1.50 (s, 6H), 0.83 (s, 9H), 0.01 (s, 6H);
1
3
C NMR (CDCl ) δ 128.75, 71.11, 45.26, 44.09, 25.78, 18.09, 13.76, −5.76;
3
Anal. Calc. for C H O Si: C, 66.61; H, 11.18. Found: C, 66.55; H, 11.08.
1
5
30
2
[1-(t-Butyldimethyl-silanyloxymethyl)
3,4-dimethyl-cyclopent-3-enyl
methoxymethyl] phos phonic acid diisopropyl ester (9): To a solution of the
cyclopentenol 8 (2.3 g, 8.50 mmol) in 5 mL of DMF was added LiI (85.36 mg,
◦
0
.64 mmol) at 25 C. Both LiOt-Bu (13.61 mL of 1.0 M solution in THF, 13.61
mmol) and a solution of diisopropyl bromomethylphosphonate (2.74 mL,
1.52 mmol) in 5 mL of DMF were slowly added to the reaction mixture and
1

New Acyclic Bis(SATE) Nucleoside Prodrugs
587
◦
stirred for 2 hours at 60 C under anhydrous conditions. The mixture was
quenched by adding water (50 mL), and the organic solvents were removed
in vacuo. The aqueous layer was extracted with EtOAc (3 × 100 mL). The
combined extracts were washed with brine, dried over MgSO , and
4
concentrated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc/hexane, 1:2) to give 9 (2.44 g, 64%) as a colorless
1
syrup: H NMR (CDCl , 300 MHz) δ 4.72 (m, 2H), 3.73 (m, 2H), 3.42 (s,
3
2
H), 3.41 (s, 2H), 2.14–1.99 (m, 4H), 1.52 (s, 6H), 1.29 (m, 12H), 0.85 (s,
13
9H), 0.00 (s, 6H); C NMR (CDCl ) δ 128.81, 70.87, 67.36, 66.30, 65.15,
3
4
5.78, 44.20, 25.87, 24.13, 18.25, 13.76, −5.46; Anal. Calc. for C H O PSi:
22
45
5
C, 58.90; H, 10.11, Found: C, 58.95; H, 10.08.
(
1-Hydroxymethyl-3,4-dimethyl-cyclopent-3-enylmethoxymethyl) phos-
phonic acid diisopropyl ester (10): To a solution of 9 (176 mg, 0.392 mmol)
in tetrahydrofuran (6 mL) was added tetrabutylammonium fluoride (TBAF)
◦
(
0.60 mL, 1.0 M solution in THF) at 0 C and stirred for 5 hours at room
temperature. The reaction mixture was concentrated in vacuo and the
residue was purified by silica gel column chromatography (EtOAc/hexane,
1
4
:1) to give 10 (108 mg, 83%) as a white solid: H NMR (CDCl , 300 MHz)
3
δ 4.68 (m, 2H), 3.69 (m, 2H), 3.54 (s, 2H), 3.47 (s, 2H), 2.12–1.99 (m, 4H),
13
1
.50 (s, 6H), 1.29 (m, 12H); C NMR (CDCl ) δ 128.68, 78.97, 71.18, 67.75,
3
6
6.79, 64.59, 45.40, 44.40, 23.98, 13.67; Anal. Calc. for C H O P: C, 57.47;
16
31
5
H, 9.34. Found: C, 57.52; H, 9.43.
Methanesulfonic acid 1-(diisopropoxy-phosphorylmethoxymethyl) 3,4
-dimethylcyclopent-3-enylmethyl ester (11): To a solution of the alcohol
1
0 (789 mg, 2.36 mmol) in anhydrous CH Cl2 (25 mL), anhydrous
2
triethylamine (0.75 mL, 5.42 mmol) and MsCl (0.22mL, 2.83 mmol) were
◦
added at 0 C. The mixture was stirred overnight at the same tempera-
ture, quenched by a cold saturated NaHCO solution (1.5 mL), and fur-
3
ther diluted with water (100 mL). The mixture was extracted with CH Cl2
2
(
100 mL × 2) and water. The combined organic layer was washed with brine,
dried over anhydrous magnesium sulfate, and filtered. The filtrate was con-
centrated under vacuum, and the residue was purified by flash silica gel
column chromatography (EtOAc/hexane, 1:1) to give 11 (749 mg, 77%) as
1
a colorless syrup: H NMR (CDCl , 300 MHz) δ 4.73 (m, 2H), 4.14 (s, 2H),
3
3
1
.73 (m, 2H), 3.49 (s, 2H), 3.03 (s, 3H), 2.18 (s, 4H), 1.56 (s, 6H), 1.34 (m,
2H); 13C NMR (CDCl ) δ 128.55, 72.84, 71.03, 67.27, 65.05, 44.20, 36.88,
3
2
4.09, 13.62; Anal. Calc. for C H O PS: C, 49.50, H, 8.06. Found: C, 49.56;
17
33
7
H, 7.93.
[
1-(6-Aminopurin-9-ylmethyl)
3,4-dimethylcyclopent-3-enylmethoxy
methyl] phosphonic acid diisopropyl ester (12): A solution of the mesylate
1
1 (453 mg, 1.10 mmol), K CO (303 mg, 2.19 mmol), 18-crown-6 (441 mg,
2 3
1
.67 mmol), and adenine (150 mg, 1.11 mmol) in dry DMF (15 mL) was
◦
stirred overnight at 90 C. The mixture was cooled to room temperature and
concentrated under vacuum. The residue was diluted with water (100 mL)

5
88
H. Li and J. H. Hong
and extracted with CH Cl (100 mL × 3). The combined organic layer was
2
2
dried over anhydrous MgSO , filtered, and concentrated under reduced
4
pressure. The residue was purified by silica gel column chromatography
(
MeOH/CH Cl , 1:10) to give compound 12 (159 mg, 32%) as a white
2 2
solid1:
H NMR (CDCl , 300 MHz) δ 8.34 (s, 1H), 8.03 (s, 1H), 4.80 (m, 2H),
3
4
1
7
7
.28 (s, 2H), 3.75 (m, 2H), 3.35 (s, 2H), 2.40–2.04 (m, 4H), 1.55 (s, 6H),
13
.36 (m, 12H); C NMR (CDCl ) δ 155.32, 152.73, 142.24, 128.68, 119.12,
3
1.07, 48.09, 45.45, 24.13, 13.66; Anal. Calc. for C H N O P: C, 55.86; H,
21
34
5
4
.59; N, 15.51. Found: C, 55.76; H, 7.44; N, 15.47.
1-(4-Amino-2-oxo-2H-pyrimidin-1-ylmethyl)
[
3,4-dimethylcyclopent-3-
enylmethoxymethyl] phosphonic acid diisopropyl ester (13): Cytosine
derivative 13 was synthesized from 11 by a similar procedure as described
◦
1
for 12. Yield 37% as a white solid: m.p. 146–148 C; H NMR (CDCl , 300
3
MHz) δ 7.33 (d, J = 7.0 Hz, 1H), 5.54 (s, J = 7.0 Hz, 1H), 4.75 (m, 2H), 4.31
(
2
1
s, 2H), 3.74 (d, J = 8.0 Hz, 2H), 3.32 (s, 2H), 2.38 (d, J = 10.2 Hz, 2H),
13
.08 (d, J = 10.0 Hz, 2H), 1.55 (s, 6H), 1.34 (m, 12H); C NMR (CDCl ) δ
3
65.23, 156.62, 145.70, 127.43, 98.32, 71.11, 48.56, 45.38, 25.49, 12.99; Anal.
Calc. for C H N O P (+ 1.0 MeOH): C, 54.89; H, 8.33; N, 9.14. Found: C,
20
34
3
5
5
4.92; H, 8.28; N, 9.17.
1-(6-Aminopurin-9-ylmethyl)
methyl] phosphonic acid (15): To a solution of the phosphonate 12 (92 mg,
[
3,4-dimethylcyclopent-3-enylmethoxy
0
2
.204 mmol) in CH CN (8 mL) was added trimethylsilyl bromide (336 mg,
.22 mmol). The mixture was heated under reflux for 15 hours and then
3
concentrated under reduced pressure. The residue was partitioned between
CH Cl (20 mL) and distilled H O (20 mL). The aqueous layer was washed
2
2
2
out with CH Cl and then freeze-dried to give target compound 15 (64 mg,
2
2
1
8
6%) as a yellowish foamy solid. UV (H O) λmax 261.5 nm (ε 14 723); H
2
NMR (DMSO-d , 300 MHz) δ 8.19 (s, 1H), 8.05 (s, 1H), 4.79 (m, 2H), 3.78
6
13
(
(
d, J = 8.0 Hz, 2H), 3.38 (s, 2H), 2.42–2.11 (m, 4H), 1.58 (s, 6H); C NMR
DMSO-d ) δ 154.95, 153.0q, 143.43, 128.32, 118.96, 69.34, 66.21, 49.86,
6
2
9.87, 13.50; Anal. Calc. for C H N O P (+ 2.0 H O): C, 44.66; H, 6.49;
15
22
5
4
2
N, 17.36. Found: C, 44.61; H, 6.52; N, 17.40.
1-(4-Amino-2-oxo-2H-pyrimidin-1-ylmethyl)-3,4-dimethylcyclopent-3-
[
enylmethoxy methyl] phosphonic acid (16): Cytosine phophonic acid
derivative 16 was synthesized from 13 by the similar procedure as described
for 15 as a yellowish foamy solid. Yield 80%, UV (H O) λ
272.0 nm (ε
2
max
1
5
432); H NMR (DMSO-d , 300 MHz) δ 7.41 (d, J = 7.0 Hz, 1H), 5.56
6
(
s, J = 7.0 Hz, 1H), 4.42 (s, 2H), 3.73 (d, J = 8.1 Hz, 2H), 3.31 (s, 2H),
2
1
.39–2.16 (m, 4H), 1.56 (s, 6H); 13C NMR (DMSO-d ) δ 165.62, 155.60,
6
46.71, 128.56, 99.81, 69.35, 66.34, 51.56, 42.38, 30.22, 13.48; Anal. Calc. for
C H N O P (+ 1.0 H O): C, 46.54; H, 6.69; N, 11.63. Found: C, 46.59; H,
14
22
3
5
2
6.72; N, 11.59.

New Acyclic Bis(SATE) Nucleoside Prodrugs
589
t-Butyl SATE phosphoester of 9-[1-(3,4-dimethylcyclopent-3-enylmeth
oxymethyl)] adenine (17): A solution of adenine phosphonic acid deriva-
tive 15 (65 mg, 0.177 mmol) and tributylamine (300 µL, 1.2 mmol) in water
(
2.0 mL) was mixed for 30 min and concentrated under reduced pressure.
The residue was thoroughly dried with anhydrous ethanol and toluene.
The resulting foamy solid was dissolved in anhydrous pyridine (15 mL)
to which thioester 14 (270 mg, 1.65 mmol) and 1-mesitylene-2-sulfonyl-
3
-nitro-1,2,4-triazole (222 mg, 0.75 mmol) were added. The mixture was
stirred for 16 hours at room temperature and quenched with tetrabuty-
lammonium bicarbonate buffer (7.5 mL, 1 M solution, pH = 8.0). The
mixture was concentrated under reduced pressure and the residue was di-
luted with water (70 mL) and extracted twice with CH Cl (70 mL). The
2
2
combined organic layer was washed with brine, dried over MgSO , filtered,
4
and evaporated. The residue was purified by silica gel column chromatogra-
phy (MeOH/Hexane/EtOAc, 0.04:4:1) to give 17 (43 mg, 37%) as a white
◦
1
solid: m.p. 116–119 C; UV (MeOH) λ
263.0 nm (ε 14 811); H NMR
max
(
3
4
DMSO-d , 300 MHz) δ 8.27 (s, 1H), 8.15 (s, 1H), 4.18 (t, J = 6.4 Hz, 4H),
6
.89 (d, J = 8.2 Hz, 2H), 3.69 (s, 2H), 3.34 (s, 2H), 3.21 (t, J = 6.3 Hz,
H), 2.32–2.11 (m, 4H), 1.57 (s, 6H), 1.25 (s, 18H); 13C NMR (DMSO-d )
6
δ 202.71, 154.44, 152.18, 149.73, 144.34, 128.40, 119.73, 79.82, 69.64, 67.47,
6
0.19, 52.15, 43.62, 30.22, 26.16, 17.0. Analysis for C H N O PS (+ 1.0
29
46
5
6
2
MeOH): C, 52.38; H, 7.33; N, 10.18; Found: C, 52.35; H, 7.36; N, 10.16.
t-Butyl SATE phosphoester of 1-[1-(3,4-dimethylcyclopent-3-enylmetho
xymethyl)] cytosine (18): The SATE prodrug of cytosine derivative 18 was
synthesized from 16 using a similar procedure as described for 17 as a white
◦
1
solid: yield 31%; m.p. 107–109 C; UV (MeOH) λ
271.0 nm (ε 5 529); H
max
NMR (CDCl , 300 MHz) δ 7.46 (d, J = 7.1 Hz, 1H), 5.61 (s, J = 7.0 Hz, 1H),
3
4
6
1
7
.22 (t, J = 6.3 Hz, 4H), 3.72 (d, J = 8.0 Hz, 2H), 3.30 (s, 2H), 3.10 (t, J =
.4 Hz, 4H), 2.96 (s, 1H), 2.31–2.13 (m, 4H), 1.57 (s, 6H), 1.23 (s,
8H; 13C NMR (CDCl ) δ 202.67, 165.66, 157.76, 145.32, 129.21, 100.03,
3
8.31, 68.54, 67.81, 55.76, 51.21, 41.79, 29.54, 26.24, 15.48,; Anal. Calc. for
C H N O PS (+ 1.0 MeOH): C, 52.27; H, 7.59; N, 6.33. Found: C, 52.29;
28
46
3
7
2
H, 7.61; N, 6.30.
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