Synthesis of phosphonate analogues of the antiviral cyclopropane nucleoside A-5021

Article abstract of DOI:10.1081/NCN-200067409

A series of phosphonate analogues of the antiviral cyclopropane nucleoside A-5021 were synthesized from (1S*, 7R*)-3,5-dioxa-4,4- diphenylbicyclo[5.1.0]octane-l-methanol by a 10-step process. In contrast to the potent antiherpetic activity of A-5021, they

Full text of DOI:10.1081/NCN-200067409

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Synthesis of Phosphonate Analogues of the Antiviral
Cyclopropane Nucleoside A-5021
Tomoyuki Onishi a , Takaaki Sekiyama a & Takashi Tsuji a
a Pharmaceutical Research Laboratories , Ajinomoto Co. Inc. , Kawasaki, Japan
Published online: 31 Aug 2006.
To cite this article: Tomoyuki Onishi , Takaaki Sekiyama & Takashi Tsuji (2005) Synthesis of Phosphonate Analogues of the
Antiviral Cyclopropane Nucleoside A-5021, Nucleosides, Nucleotides and Nucleic Acids, 24:8, 1187-1197, DOI: 10.1081/
NCN-200067409
To link to this article: http://dx.doi.org/10.1081/NCN-200067409
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (8):1187–1197, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200067409
SYNTHESIS OF PHOSPHONATE ANALOGUES OF THE ANTIVIRAL
CYCLOPROPANE NUCLEOSIDE A-5021
5
Tomoyuki Onishi, Takaaki Sekiyama, and Takashi Tsuji
Pharmaceutical
Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan
5
A series of phosphonate analogues of the antiviral cyclopropane nucleoside A-5021 were synthesized
*
*
from (1S , 7R )-3,5-dioxa-4,4-diphenylbicyclo[5.1.0]octane-l-methanol by a 10-step process. In con-
trast to the potent antiherpetic activity of A-5021, they were all devoid of antiviral activity.
Keywords Phosphate analogues, A-5021
INTRODUCTION
Phosphonate derivatives of antiviral nucleosides have been attractive targets as
potential antiviral agents, since they do not require mono-phosphorylation, the first
step in the activation of antiviral nucleosides. Among these, the R-enantiomer of
ganciclovir phosphonate (SR-3773, 1) (Figure 1) has potent and selective in vitro
activity against human cytomegalovirus (HCMV)[1,2] and in comparative studies is
more effective than ganciclovir against murine cytomegalovirus (MCMV) infections
in healthy and immunosuppressed mice.[3] Acyclovir phosphonate 2 also shows
potent in vitro activity against HCMV and MCMV, whereas 2 is less potent than
ganciclovir.[3]
We previously reported the synthesis and antiviral activity of (1S,2R)-9-[[1,2-
bis(hydroxymethyl)-cyclopropan-l-yl]methyl]guanine (A-5021, 3) (Figure 2).[4–6] A-
5021 has a unique structure with two asymmetric centers on the cyclopropane ring
as an acyclosugar moiety. A-5021 shows extremely potent antiherpetic activity
against herpes simplex virus (HSV) and varicella zoster virus, and has a greater
therapeutic effect than acyclovir in animal models.[7] Since HCMV lacks viral
thymidine kinase, which is responsible for the activation of A-5021,[8] we expected
that the phosphonate analogues of A-5021 may have better anti-HCMV activity and
Received 22 November 2004, accepted 22 March 2005.
Address correspondence to Tomoyuki Onishi, Pharmaceutical Research Laboratories, Ajinomoto Co. Inc.,
1-1 Suzuki-cho, Kawasaki 210-8681, Japan; E-mail: tomoyuki_onishi@ajinomoto.com
1187
Order reprints of this article at www.copyright.rightslink.com

1188
T. Onishi, T. Sekiyama, and T. Tsuji
FIGURE 1
started to explore their synthesis. We report here the synthesis of phosphonate
analogues of A-5021 and their antiviral activities.
RESULTS AND DISCUSSION
Since A-5021 is monophosphorylated at the 2’-site of cyclopropane by viral
thymidine kinases,[8] compound 4 (Figure 2), in which the hydroxymethyl group of
A-5021 at the 2’-site is substituted by a (dihydroxyphosphinyl)ethyl group, was
*
*
synthesized as the primary target. (1S ,7R )-3,5-Dioxa-4,4-diphenylbicyclo[5.1.0]oc-
tane-l-methanol 5, a useful intermediate for the synthesis of antiviral cyclopropane
nucleosides, was used as a starting material (Scheme 1).[9] To obtain both
*
*
*
*
(1R ,2R )- and (1R ,2S )-2-(dihydroxyphosphinyl)ethyl-1-hydroxymethylcyclopro-
pane regioisomers, 1,1-dihydroxycyclopropane 9 was used as a key synthetic
intermediate. In the presence of a catalytic amount of p-TsOH, transacetalization of
[1,3]-dioxepane 5 proceeded to give [1,3]-dioxane 6 in 43% yield. Compounds 5 and
6 could be separated by silica gel column chromatography. A diethoxyphosphinyl
group was successfully introduced by Swern oxidation of 6 followed by a Horner-
Wittig reaction.[10–12] Both reactions proceeded with good yields (87% and 82%,
respectively) to give olefin 8. Catalytic hydrogenation of 8 in the presence of acetic
acid gave 1,1-dihydroxycyclopropane 9 in 64% yield.
Next, 9 was treated with 1 equiv. of benzoyl chloride in the presence of
triethylamine to give a mixture of monobenzoyl esters 10 and 11 in 47% yield
(Scheme 2). Although 10 and 11 could not be separated by normal-phase silica gel
FIGURE 2

Synthesis of Phosphonate Analogues
1189
SCHEME 1
column chromatography, these isomers could be separated by reversed-phase C18
silica gel column chromatography eluting with 30% acetonitrile to give 10 and 11 in
respective yields of 19% and 9%. The structure of each isomer could be clearly
determined by NOESY.
To prepare the phosphonate analogue of A-5021 4, alcohol 11 was converted to
a bromide by treatment with CBr4-Ph3P in the presence of triethylamine, and the
resulting bromide was used without further isolation for the alkylation of 2-amino-6-
benzyloxypurine at the 9-position to give fully protected phosphonate 12 in 33%
yield from 11 (Scheme 3). Removal of the protecting groups by a three-step
sequence gave the desired product 4. The adenine analogue 14 was also
*
*
synthesized using adenine instead of 2-amino-6-benzyloxypurine. The (1R ,2S )-2-
(dihydroxyphosphinyl)ethyl-1-hydroxymethylcyclopropane regioisomers 16 and 18
were synthesized from 10 using the same method.
The antiherpetic activities of 4, 14, 16, and 18 were measured by a growth-
inhibition assay against HCMV AD169 strain[5] and by a quantitative CPE
reduction assay against HSV-1 Tomioka strain;[4] however, no inhibitory activity
was observed at up to 100 mg/mL. They also showed no cytotoxic effect in Vero
cells up to 100 mg/mL. Anti-HIV activity was also evaluated,[13] but they were
devoid of such activity. Since phosphonates are not ‘‘perfect mimics’’ for mono-
phosphates, the steps required for virus inhibition, either further phosphorylation or
DNA polymerase inhibition, might be impaired in these compounds. Poor cellular
uptake of the phosphonate analogues of A-5021 would be another possible reason
for the apparent lack of activity.[14]
SCHEME 2

1190
T. Onishi, T. Sekiyama, and T. Tsuji
SCHEME 3
EXPERIMENTAL SECTION
The reagents used were the highest quality available commercially. Unless
otherwise noted, organic extracts were dried over anhydrous Na2SO4 and
1
temperature refers to the temperature of the bath. H-NMR and NOESY spectra
were recorded with a Varian XL-300 300-MHz and a JEOL JNM-GX-400 400-MHz
spectrometer, using tetramethylsilane as an internal standard. Mass spectra were
recorded on a JEOL JMS-DX300 spectrophotometer, and accurate masses were
measured on a JEOL JMS-HX110 spectrometer. Silica gel column chromatography
was conducted on silica gel 60 (70–230 mesh; Merck Art. no. 7734). Preparative
reversed-phase column chromatography was conducted on a Merck LiChroprep
RP-18 column (40–63 mm).
5,7-Dioxa-6,6-diphenyl-1-hydroxymethylspiro[2.5]octane (6).
*
*
To a solution of (1S ,7R )-3,5-dioxa-4,4-diphenylbicyclo[5.1.0]octane-1-methanol
(10.00 g, 33.7 mmol) in dry THF (67.4 mL) was added p-toluenesulfonic acid
monohydrate (321 mg, 1.69 mmol). After the solution was stirred for 12 h at room
temperature, a saturated aqueous solution of sodium hydrogen carbonate was
added, and the resulting mixture was extracted with ethyl acetate. The organic layer
was dried over anhydrous sodium sulfate, and the solvent was then removed under
reduced pressure. The residue was purified by silica gel chromatography using a
step gradient of 20% and 25% ethyl acetate in hexane, to give 6 (4.32 g, 43%) as a white
1
solid. H-NMR (CDCl3) d 0.39 (t, J = 5.5 Hz, 1H), 0.66 (dd, J = 5.5, 8.7 Hz, 1H),
1.33–1.43 (m, 1H), 2.15–2.25 (m, 1H), 3.37 (d, J = 11.7 Hz, 1H), 3.41 (dd, J = 1.8,
11.4 Hz, 1H), 3.78 (dd, J = 2.1, 12.0 Hz, 1H), 3.88–3.99 (m, 1H), 4.17 (d, J = 11.4 Hz,
1H), 4.21 (d, J = 12.0 Hz, 1H), 7.18–7.59 (m, 10H); FAB MASS, 297 (MH+).
5,7-Dioxa-6,6-diphenyl-1-formylspiro[2.5]octane (7). To a so-
lution of oxalyl chloride (2.14 g, 16.9 mmol) in dichloromethane (39 mL) was

Synthesis of Phosphonate Analogues
1191
added a solution of dimethylsulfoxide (2.65 g, 33.9 mmol) in dichloromethane (8.4
mL) at À50°C. After the solution was stirred for 2 min, a solution of 6 (4.56 g, 15.4
mmol) in dichloromethane (16 mL) was added. After this solution was stirred for 15
min, triethylamine (10.8 mL) was added. After this mixture was stirred for 5 min, it
was allowed to warm to room temperature. To the solution was added water, and
the resulting mixture was extracted with dichloromethane. The organic layer was
washed with a saturated aqueous solution of sodium chloride and dried over
anhydrous sodium sulfate, and then the solvent was removed under reduced
pressure. The residue was purified by silica gel chromatography using a gradient of
1
hexane to 9% ethyl acetate in hexane, to give 7 (3.95 g, 87%) as a white solid. H-
NMR (CDCl3) d 1.11 (dd, J = 5.4, 8.1 Hz, 1H), 1.49 (t, J = 5.4 Hz, 1H), 2.09 (ddd,
J = 3.2, 5.4, 8.1 Hz, 1H), 3.68 (dd, J = 1.6, 11.6 Hz, 1H), 3.99 (d, J = 11.6 Hz, 1H),
4.02 (dd, J = 1.6, 11.8 Hz, 1H), 4.14 (d, J = 11.8 Hz, 1H), 7.21–7.41 (m, 6H), 7.46–
7.58 (m, 4H), 9.73 (d, J = 3.2 Hz); FAB MASS, 295 (MH+).
1-(E)-(Diethoxyphosphinyl)ethenyl-5,7-dioxa-6,6-diphenyl-
spiro[2.5]octane (8). To a solution of tetraethyl methylenediphosphonate
(1.69 g, 5.88 mmol) in dry THF (7.8 ml) was added a solution of 1.6 M n-
butyllithium in hexane (3.68 mL, 5.88 mmol) at À78°C. After this solution was
stirred for 1 h, a solution of 7 (1.73 g, 5.88 mmol) in dry THF (7.8 mL) was added.
After this mixture was stirred for an additional 1 h, a saturated aqueous solution of
ammonium chloride was added. The resulting mixture was extracted with ethyl
acetate, and the organic layer was dried over anhydrous sodium sulfate. The
solvent was removed under reduced pressure, and the residue was purified by
silica gel chromatography using a step gradient of 17% to 67% ethyl acetate in
hexane, to give 8 (2.08 g, 82%) as a colorless oil. 1H-NMR (CDCl3) d 0.90 (t, J = 5.6
Hz, 1H), 1.08 (dd, J = 5.6, 8.4 Hz, 1H), 1.29 (t, J = 7.1 Hz, 3H), 1.31 (t, J = 7.1 Hz,
3H), 1.60–1.72 (m, 1H), 3.75 (d, J = 11.6 Hz, 1H), 3.83 (d, J = 11.6 Hz, 1H), 3.98
(s, 2H), 3.99–4.10 (m, 4H), 5.75 (ddd, J = 0.6, 16.8, 19.5 Hz, 1H), 6.46 (ddd,
J = 9.6, 16.8, 26.4 Hz, 1H), 7.22–7.38 (m, 6H), 7.45–7.55 (m, 4H); FAB MASS,
429 (MH+).
1,1-Bis(hydroxymethyl)-2-[(diethoxyphosphinyl)ethyl]cyclo-
propane (9). To a solution of 8 (2.07 g, 4.83 mmol) in ethanol (48.3 mL) was
added palladium carbon (10%) (0.1 g) and the mixture was stirred in a hydrogen
atmosphere pressurizing to 20 PSI for 13 h. To this mixture was added acetic acid
(2.4 mL) and the mixture was stirred for an additional 4.5 h. The mixture was
filtered and the filtrate was concentrated under reduced pressure. The residue was
purified by silica gel chromatography using a gradient of dichloromethane to 5%
1
methanol in dichloromethane, to give 9 (819 mg, 64%) as a colorless oil. H-NMR
(CD3OD) d 0.28 (t, J = 5.0 Hz, 1H), 0.68 (dd, J = 5.0, 8.4 Hz, 1H), 0.90–1.00 (m,
1H), 1.37 (t, J = 7.1 Hz, 6H), 1.56–2.12 (m, 4H), 3.45 (d, J = 11.1 Hz, 1H), 3.55 (d,
J = 11.1 Hz, 1H), 3.58 (d, J = 11.6 Hz, 1H), 3.83 (d, J = 11.6 Hz, 1H), 4.07–4.18 (m,
4H); ESI MASS, 289.5 (M + Na+).

1192
T. Onishi, T. Sekiyama, and T. Tsuji
*
*
(1R ,2R )-1-Benzoyloxymethyl-2-(diethoxyphosphinyl)ethyl-
*
*
1-hydroxymethyl-cyclopropane (10) and (1R ,2S )-1-benzoyloxy-
methyl-2-(diethoxyphosphinyl)ethyl-1-hydroxymethyl-cyclopro-
pane (11). To a mixture of 9 (605 mg, 2.27 mmol) and triethylamine (460 mg,
4.55 mmol) in dichloromethane (11 mL) was added a solution of benzoyl chloride
(320 mg, 2.27 mmol) in dichloromethane (11 mL) at room temperature. After this
mixture was stirred for 1.5 h, a saturated aqueous solution of sodium hydrogen
carbonate was added. The resulting mixture was extracted with dichloromethane,
and the organic layer was dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the residue was purified by silica gel
chromatography to give a mixture of 10 and 11 (386 mg, 47%). Isomers 10 and 11
were isolated by chromatography on a column of reversed-phase C18 silica gel
eluting with 30% acetonitrile in water, to give 10 (157 mg, 19%) and 11 (73.1 mg, 9%).
10 (colorless oil); 1H-NMR (CDCl3) d 0.31 (t, J = 5.6 Hz, 1H), 0.84 (dd, J = 5.6,
8.7 Hz, 1H), 0.98–1.09 (m, 1H), 1.32 (t, J = 6.9 Hz, 6H), 1.65–1.99 (m, 4H), 2.86 (t,
J = 6.2 Hz, 1H), 3.50 (dd, J = 6.2, 12.2 Hz, 1H), 3.90 (dd, J = 6.2, 12.2 Hz, 1H),
4.03–4.16 (m, 5H), 4.54 (dd, J = 1.1, 11.6 Hz, 1H), 7.41–7.48 (m, 2H), 7.54–7.60
(m, 1H), 8.02–8.08 (m, 2H); ESI MASS, 371.3 (MH+).
1
11 (colorless solid); H-NMR (CDCl3) d 0.45 (t, J = 5.3 Hz, 1H), 0.76 (dd,
J = 5.3, 8.4 Hz, 1H), 1.03–1.14 (m, 1H), 1.29 (t, J = 7.1 Hz, 3H), 1.30 (t, J = 7.1 Hz,
3H), 1.47–2.01 (m, 4H), 3.30 (d, J = 11.7 Hz, 1H), 3.65 (d, J = 11.7 Hz, 1H), 3.98–
4.15 (m, 4H), 4.41 (d, J = 11.7 Hz, 1H), 4.54 (d, J = 11.7 Hz, 1H), 7.39–7.44 (m,
2H), 7.51–7.58 (m, 1H), 8.02–8.08 (m, 2H); ESI MASS, 371.3 (MH+).
Determination of the Structures of 10 and 11 by NOESY
In 10, NOE was observed between the proton at position 2 of the cyclopropane
and the methylene proton of the benzoyloxymethyl group at position 1, between
the proton at position 3(a) of cyclopropane and the methylene proton of the
benzoyloxymethyl group at position 1, and between the proton at position 3(b) of
cyclopropane and the methylene proton of the hydroxymethyl group at position 1.
On the other hand, in 11, NOE was observed between the proton at position 3(b)
of cyclopropane and the methylene proton of the benzoyloxymethyl group at
position 1.
*
*
2-Amino-6-benzyloxy-9-[(1R ,2R )-1-benzoyloxymethyl-2-
[(diethoxyphosphinyl)-ethyl]cyclopropan-1-yl]methylpurine
(12). To a mixture of 11 (36.6 mg, 0.099 mmol), triethylamine (5 mg, 0.049
mmol) and triphenylphosphine (47 mg, 0.178 mmol) in dichloromethane (1 mL)
was added carbon tetrabromide (59 mg, 0.178 mmol) at 0°C. After this mixture
was stirred for 1 h at 0°C, phosphate buffer (pH = 7) was added, and the resulting
mixture was extracted with dichloromethane. The organic layer was dried over
anhydrous sodium sulfate, and the solvent was removed under reduced pressure.

Synthesis of Phosphonate Analogues
1193
The residue was dissolved in dry DMF (1 mL), and to the solution were added 2-
amino-6-benzyloxypurine (23.8 mg, 0.099 mmol) and potassium carbonate (13.6
mg, 0.099 mmol). After this mixture was stirred for 12 h at room temperature,
insoluble substances were removed by filtration. The filtrate was concentrated
under reduced pressure, and the residue was purified by preparative thin-layer
chromatography, eluting with 10% methanol in dichloromethane, to give 12 (19.5
1
mg, 33%) as a colorless solid. H-NMR (CD3OD) d 0.65 (t, J = 5.6 Hz, 1H), 1.14
(dd, J = 5.6, 8.7 Hz, 1H), 1.21 (t, J = 7.4 Hz, 3H), 1.24 (t, J = 7.4 Hz, 1H), 1.54–1.73
(m, 2H), 1.79–2.12 (m, 3H), 3.88–4.02 (m, 5H), 4.12 (d, J = 12.3 Hz, 1H), 4.46 (d,
J = 13.5 Hz, 1H), 4.74 (d, J = 13.5 Hz, 1H), 5.38 (d, J = 12.3 Hz, 1H), 5.48 (d,
J = 12.3 Hz, 1H), 7.30–7.45 (m, 5H), 7.47–7.60 (m, 5H), 8.00 (s, 1H); ESI MASS,
594.5 (MH+).
*
*
9-[(1R ,2R )-2-(Dihydroxyphosphinyl)ethyl-1-hydroxyme-
thylcyclopropan-1-yl]-methylguanine (4). To a solution of 12 (19.5
mg, 0.033 mmol) in dry DMF (0.4 mL) was added trimethylsilyl bromide (75.9 mL,
0.575 mmol) at room temperature. After this mixture was stirred for 43 h, the
solvent was removed, coevaporating three times with methanol. The residue was
dissolved in 1 M NaOH (0.7 mL). After this mixture was stirred for 2 h, 2 M HCl
(0.7 mL) was added. After this mixture was stirred for an additional 20 min, 1 M
NaOH (0.7 mL) was added. The mixture was purified by chromatography on a
column of reversed-phase C18 silica gel eluting with water to give 4 (11.3 mg, 100%)
1
as a white solid. H-NMR (d6-DMSO) d 0.16 (t, J = 5.1 Hz, 1H), 0.85 (dd, J = 5.1,
8.4 Hz, 1H), 1.03–1.17 (m, 1H), 1.47–1.64 (m, 4H), 3.17 (d, J = 12.0 Hz, 1H), 3.36
(d, J = 12.0 Hz, 1H), 3.72 (d, J = 14.1 Hz, 1H), 4.08 (d, J = 14.1 Hz, 1H), 6.61 (bs,
2H), 7.86 (s, 1H), 10.74 (bs, 1H); 13C-NMR (d6-DMSO) d 14.7, 22.2 [ J(13C,31P) = 3.7
Hz], 23.3 [ J(13C,31P) = 19.2 Hz], 27.0, 27.8 [ J(13C,31P) = 134.7 Hz], 47.8, 60.1,
137.7, 151.1, 153.7, 156.3; 31P-NMR (d6-DMSO) d 26.1; UV lmax (0.1 M HCl) 281 sh
nm (e 7400), 255 (e 11000); HRMS calcd for C12H19N5O5P (MH+) 344.1124,
found 344.1129.
*
*
9-[(1R ,2R )-1-Benzoyloxymethyl-2-[(diethoxyphosphinyl)-
ethyl]cyclopropan-l-yl]methyladenine (13). To a mixture of 11
(36.6 mg, 0.099 mmol), triethylamine (5 mg, 0.049 mmol) and triphenylphosphine
(47 mg, 0.178 mmol) in dichloromethane (1 mL) was added carbon tetrabromide
(59 mg, 0.178 mmol). After this mixture was stirred for 1 h at 0°C, phosphate buffer
(pH = 7) was added, and the resulting mixture was extracted with dichloro-
methane. The organic layer was dried over anhydrous sodium sulfate, and the
solvent was removed under reduced pressure. The residue was dissolved in dry
DMF (0.5 mL), and the solution was added to a suspension of adenine (13.3 mg,
0.099 mmol) and sodium hydride (60% oil dispersion, 3.9 mg, 0.099 mmol) in dry
DMF (0.5 mL). After this mixture was stirred for 12 h at 60°C, insoluble substances
were removed by filtration. The filtrate was concentrated under reduced pressure,

1194
T. Onishi, T. Sekiyama, and T. Tsuji
and the residue was purified by preparative thin-layer chromatography, eluting with
1
10% methanol in dichloromethane, to give 13 (24.8 mg, 52%) as a white solid. H-
NMR (CD3OD) d 0.69 (t, J = 5.4 Hz, 1H), 1.17–1.28 (m, 7H), 1.58–1.73 (m, 2H),
1.78–2.10 (m, 3H), 3.91–4.04 (m, 4H), 4.14 (d, J = 12.6 Hz, 1H), 4.16 (d, J = 14.6
Hz, 1H), 4.55 (d, J = 14.6 Hz, 1H), 4.72 (d, J = 12.6 Hz, 1H), 7.37–7.45 (m, 2H),
7.51–7.61 (m, 3H), 8.11 (s, 1H), 8.32 (s, 1H); ESI MASS, 488.3 (MH+).
*
*
9-[(1R ,2R )-2-(Dihydroxyphosphinyl)ethyl-1-hydroxyme-
thylcyclopropan-1-yl]-methyladenine (14). To a solution of 13 (24.8
mg, 0.051 mmol) in dry DMF (0.5 mL) was added trimethylsilyl bromide (117.5 mL,
0.890 mmol) at room temperature. After this mixture was stirred for 41 h, the
solvent was removed, coevaporating three times with methanol. The residue was
dissolved in 1 M NaOH (1.0 mL). After this mixture was stirred for 2 h, 2 M HCl
(0.5 mL) was added. The mixture was purified by chromatography on a column of
reversed-phase C18 silica gel using a gradient of water to 5% methanol in water, to
1
give 14 (14.8 mg, 89%) as a white solid. H-NMR (d6-DMSO) d 0.16 (t, J = 4.9 Hz,
1H), 0.88 (dd, J = 4.9, 8.6 Hz, 1H), 1.08–1.18 (m, 1H), 1.46–1.60 (m, 4H), 3.17 (d,
J = 12.2 Hz, 1H), 3.34 (d, J = 12.2 Hz, 1H), 3.91 (d, J = 14.3 Hz, 1H), 4.26 (d,
J = 14.3 Hz, 1H), 7.21 (bs, 2H), 8.11 (s, 1H), 8.13 (s, 1H); 13C-NMR (d6-DMSO) d
14.8, 22.2 [ J(13C,31P) = 3.7 Hz], 23.5 [ J(13C,31P) = 20.2 Hz], 27.1, 27.9
[ J(13C,31P) = 135.6 Hz], 47.9, 60.1, 118.4, 141.1, 149.8, 152.3, 155.9; 31P-NMR (d6-
DMSO) d 26.0; UV lmax (0.1 M HCl) 260 sh nm (e 14,400); HRMS calcd for
C12H19N5O4P (MH+) 328.1175, found 328.1177.
*
*
2-Amino-6-benzyloxy-9-[(1R ,2S )-1-benzoyloxymethyl-2-
[(diethoxyphosphinyl)-ethyl]cyclopropan-1-yl]methylpurine
(15). To a mixture of 10 (40.3 mg, 0.109 mmol), triethylamine (5.5 mg, 0.055
mmol) and triphenylphosphine (51.5 mg, 0.196 mmol) in dichloromethane (1.1
mL), was added carbon tetrabromide (65 mg, 0.196 mmol) at 0°C. After this
mixture was stirred for 1.5 h at 0°C, phosphate buffer (pH = 7) was added, and the
resulting mixture was extracted with dichloromethane. The organic layer was dried
over anhydrous sodium sulfate, and the solvent was removed under reduced
pressure. The residue was dissolved in dry DMF (1.1 mL), and to the solution were
added 2-amino-6-benzyloxypurine (26.2 mg, 0.109 mmol) and potassium carbonate
(15.0 mg, 0.109 mmol). After this solution was stirred for 15 h at room temperature,
insoluble substances were removed by filtration. The filtrate was concentrated
under reduced pressure, and the residue was purified by preparative thin-layer
chromatography, eluting with 10% methanol in dichloromethane, to give 15 (17.2
1
mg, 27%) as a colorless solid. H-NMR (CD3OD) d 0.94 (t, J = 5.5 Hz, 1H), 1.00
(dd, J = 5.5, 8.6 Hz, 1H), 1.25–1.41 (m, 1H), 1.37 (t, J = 7.2 Hz, 6H), 1.84–2.12 (m,
4H), 4.07–4.25 (m, 7H), 4.63 (d, J = 15.0 Hz, 1H), 5.42 (d, J = 12.3 Hz, 1H), 5.47 (d,
J = 12.3 Hz, 1H), 7.32–7.44 (m, 5H), 7.49–7.57 (m, 3H), 7.68–7.74 (m, 2H), 7.98 (s,
1H); ESI MASS, 594.5 (MH+).

Synthesis of Phosphonate Analogues
1195
*
*
9-[(1R ,2S )-2-(Dihydroxyphosphinyl)ethyl-1-hydroxyme-
thylcyclopropan-1-yl]-methylguanine (16). To a solution of 15
(17.2 mg, 0.029 mmol) in dry DMF (0.3 mL) was added trimethylsilyl bromide
(66.9 mL, 0.507 mmol) at room temperature. After the solution was stirred for 47 h,
the solvent was removed, coevaporating three times with methanol. The residue
was dissolved in 1 M NaOH (0.7 mL). After this mixture was stirred for 3 h, 2 M
HCl (0.7 mL) was added. After this mixture was stirred for an additional 30 min, 1
M NaOH (0.7 mL) was added. The mixture was purified by chromatography on a
column of reversed-phase C18 silica gel using a gradient of water to 5% methanol in
water, to give 16 (10.0 mg, 100%) as a white solid. 1H-NMR (d6-DMSO) d 0.48 (dd,
J = 4.8, 8.5 Hz, 1H), 0.54 (t, J = 4.8 Hz, 1H), 0.93–0.99 (m, 1H), 1.52–1.72 (m, 4H),
2.76 (d, J = 11.2 Hz, 1H), 3.17 (d, J = 11.2 Hz, 1H), 3.72 (d, J = 14.2 Hz, 1H), 4.33
(d, J = 14.2 Hz, 1H), 6.48 (bs, 2H), 7.65 (s, 1H), 10.54 (bs, 1H); 31P-NMR (d6-
DMSO) d 26.3; UV lmax (0.1 M HCl) 281 sh nm (e 6900), 255 (e 10200); HRMS
calcd for C12H19N5O5P (MH+) 344.1124, found 344.1103.
*
*
9-[(1R ,2S )-1-Benzoyloxymethyl-2-[(diethoxyphosphinyl)-
ethyl]cyclopropan-l-yl]-methyladenine (17). To a mixture of 10 (40.3
mg, 0.109 mmol), triethylamine (5.5 mg, 0.055 mmol) and triphenylphosphine (51.5
mg, 0.196 mmol) in dichloromethane (1.1 mL) was added carbon tetrabromide (65
mg, 0.196 mmol) at 0°C. After the mixture was stirred for 1.5 h, phosphate buffer
(pH = 7) was added, and the resulting mixture was extracted with dichloro-
methane. The organic layer was dried over anhydrous sodium sulfate, and the
solvent was removed under reduced pressure. The residue was dissolved in dry
DMF (0.5 mL), and the solution was added to a suspension of adenine (14.7 mg,
0.109 mmol) and sodium hydride (60% oil dispersion, 4.35 mg, 0.319 mmol) in dry
DMF (0.6 mL). After this mixture was stirred for 15 h at 60°C, insoluble substances
were removed by filtration. The filtrate was concentrated under reduced pressure,
and the residue was purified by preparative thin-layer chromatography, eluting with
10% methanol in dichloromethane, to give 17 (3.1 mg, 6%) as a white solid. 1H-NMR
(CD3OD) d 0.98–1.07 (m, 2H), 1.32–1.41 (m, 1H), 1.38 (t, J = 7.2 Hz, 6H), 1.92–
2.13 (m, 4H), 4.10–4.23 (m, 6H), 4.28 (d, J = 14.6 Hz, 1H), 4.81 (d, J = 14.6 Hz,
1H), 7.39–7.47 (m, 2H), 7.49–7.63 (m, 1H), 7.63–7.73 (m, 2H), 8.13 (s, 1H), 8.29
(s, 1H); ESI MASS, 488.4 (MH+).
*
*
9-[(1R ,2S )-2-(Dihydroxyphosphinyl)ethyl-l-hydroxymethyl-
cyclopropan-1-yl]-methyladenine (18). To a solution of 17 (3.1 mg,
0.0064 mmol) in dry DMF (63.6 mL) was added trimethylsilyl bromide (14.7 mL,
0.111 mmol) at room temperature. After this mixture was stirred for 65 h, the
solvent was removed, coevaporating three times with methanol. The residue was
dissolved in 1 M NaOH (1.0 mL). After this mixture was stirred for 2 h, 2 M HCl
(0.5 mL) was added. The mixture was purified by chromatography on a column of
reversed-phase C18 silica gel using a gradient of water to 5% methanol in water, to

1196
T. Onishi, T. Sekiyama, and T. Tsuji
give 18 (2.1 mg, 100%) as a white solid. 1H-NMR (D2O) d 0.58–0.78 (m, 2H), 1.04–
1.11 (m, 1H), 1.65–1.85 (m, 4H), 3.07 (d, J = 12.0 Hz, 1H), 3.36 (d, J = 12.0 Hz,
1H), 3.99 (d, J = 14.8 Hz, 1H), 4.45 (d, J = 14.8 Hz, 1H), 7.98 (s, 1H), 8.32 (s, 1H);
31P-NMR (d6-DMSO) d 26.5; HRMS calcd for C12H19N5O4P (MH+) 328.1175,
found 328.1190.
Antiviral Activity
A quantitative CPE reduction assay against HSV-I was performed using the
neutral red dye uptake method as described previously.[4] A growth-inhibition assay
of MRC-5 cells against HCMV was performed as described previously.[5] An anti-
HIV assay was also performed as described previously.[12]
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