Design and synthesis of novel threosyl-5′-deoxyphosphonic acid purine analogues as potent anti-HIV agents

Article abstract of DOI:10.1080/15257770.2012.670349

The discovery of threosyl phosphonate nucleoside (PMDTA, EC₅₀ = 2.53 μM) as a potent anti-HIV agent has led to the synthesis and biological evaluation of 5′-deoxyversions of threosyl phosphonate nucleosides from 1,4-dihydroxy-2-butene. The synthesized nucleoside phosphonic acid analogues 14 and 19 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 14 exhibits moderate in vitro anti-HIV-1 activity (EC₅₀ = 12.6 μM). Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2012.670349

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Design and Synthesis of Novel
Threosyl-5′- Deoxyphosphonic Acid
Purine Analogues as Potent Anti-Hiv
Agents
Lian Jin Liu a , Eunae Kim a & Joon Hee Hong a
a BK-21 Project Team, College of Pharmacy, Chosun University,
Kwangju, Republic of Korea
Published online: 12 Apr 2012.
To cite this article: Lian Jin Liu , Eunae Kim & Joon Hee Hong (2012): Design and Synthesis of
Novel Threosyl-5′- Deoxyphosphonic Acid Purine Analogues as Potent Anti-Hiv Agents, Nucleosides,
Nucleotides and Nucleic Acids, 31:5, 411-422
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Nucleosides, Nucleotides and Nucleic Acids, 31:411–422, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.670349
DESIGN AND SYNTHESIS OF NOVEL THREOSYL-5ꢀ-
DEOXYPHOSPHONIC ACID PURINE ANALOGUES AS POTENT
ANTI-HIV AGENTS
Lian Jin Liu, Eunae Kim, and Joon Hee Hong
BK-21 Project Team, College of Pharmacy, Chosun University, Kwangju,
Republic of Korea
The discovery of threosyl phosphonate nucleoside (PMDTA, EC50 = 2.53 µM) as a potent
2
anti-HIV agent has led to the synthesis and biological evaluation of 5 ꢁ-deoxyversions of threosyl
phosphonate nucleosides from 1,4-dihydroxy-2-butene. The synthesized nucleoside phosphonic acid
analogues 14 and 19 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue
14 exhibits moderate in vitro anti-HIV-1 activity (EC50 = 12.6 µM).
Keywords Antiviral agent; threosyl nucleoside phosphonic acid; Vorbruggen reaction
INTRODUCTION
Much attention has been paid to unusual nucleosides, as modified nu-
cleosides were reported to be the promising anti-human immunodeficiency
virus (HIV) and anti-hepatitis B virus (HBV) agents. Among these com-
pounds, threose nucleosides,[1] such as PMDTA (1) and PMDTT (2), have
been previously synthesized (Figure 1) because they can be assembled from
natural precursor molecules.[2] It has been demonstrated that threose nu-
cleic acids (TNA) form duplex with DNA and RNA of thermal stability, sim-
ilar to that of the natural nucleic acid association. Triphosphates of threose
nucleosides are accepted as substrates by several polymerases, and they can
be enzymatically incorporated into DNA.[3] These nucleosides are accepted
as substitutes for ribonucleosides in the catalytic site of a hammerhead ri-
bozyme, although the catalytic efficiency of the ribozyme is significantly
reduced.[4] The phosphonoalkoxy group of the threose nucleoside phos-
phonates is bound at the 3ꢁ-position. It brings the phosphorus atom and
the nucleobase closer to each other than in previously synthesized furanose
Received 4 December 2011; accepted 23 February 2012.
This study was supported by a research grant of Chosun University, 2012.
Address correspondence to Joon Hee Hong, College of Pharmacy, Chosun University, Kwangju
501-759, Republic of Korea. E-mail: hongjh@chosun.ac.kr
411

412
L. J. Liu et al.
FIGURE 1 Structures of nucleoside phosphonic acid as potent antiviral agents.
nucleoside phosphonates, where the phosphonate group is bound to the
primary hydroxyl group at the 4ꢁ-position.
In the literature, several 5ꢁ-phosphate isosteres have been adopted to
prepare nucleoside phosphonates. As shown in Figure 1, compounds 3[5]
and 4[6] are simple 5ꢁ-deoxynucleoside phosphonates, in which the 5ꢁ-oxygen
of a nucleoside phosphate is either removed or replaced by methylene group
(Figure 1). All of the resultant phosphonates mimic the overall shape and
geometry of a nucleoside monophosphate.
The phosphonate has certain advantages over its phosphate counter-
part, as it is metabolically stable because its phosphorus–carbon bond is not
susceptible to hydrolytic cleavage.[7] Moreover, a phosphonate nucleoside
analogue can skip the requisite first phosphorylation, which is a crucial step
for the activation of nucleosides. This is frequently a limiting event in the
phosphorylation sequence, which ultimately leads to triphosphates.[8] Like a
nucleoside monophosphate, a nucleoside phosphonate can be further phos-
phorylated by cellular nucleotide kinase. The spacial location of the carbon
atom, namely the β-position from the phosphorus atom in the nucleoside
analogue, has been demonstrated to play a critical role for antiviral activ-
ity.[9] These atoms for antiviral activity may be attributed to the increased
binding capacity of phosphonate analogues to target enzymes.[10]
Stimulated by these findings that threosyl nucleoside analogues and
5ꢁ-deoxynucleoside phosphonates have excellent biological activities, we
sought to synthesize a novel class of nucleosides comprising threosyl-5ꢁ-
deoxynucleoside phosphonic acid analogues, and evaluated their antiviral
activity against various viruses.

Threosyl 5 ꢁ-deoxyphosphonic Acid Purine Analogues
413
SCHEME 1 Synthesis of thresyl-5ꢁ-deoxyphosphonate adenine.
As depicted in Scheme 1, the target compounds were prepared from
1,4-dihydroxy-2-butene.[11] The lactone functional group of 7 was prepared
via deprotective cyclization from 6, which was reduced using DIBAL-H in
toluene at −78◦C to give 59% yield of lactol 8 as two-step yield. The lactol
8 was acetylated in pyridine to furnish the key intermediate 9 as glycosyl
donor (Scheme 1). The synthesis of adenine nucleoside was carried out by
condensation of compound 9 with silylated 6-chloropurine using TMSOTf
as a catalyst in DCE to give protected 6-chloropurine derivative 10a and 10b.
A complete NOE study allowed an unambiguous determination of their re-
spective stereochemistry (Figure 2). For compound 10b, strong NOE (1.2%)

414
L. J. Liu et al.
FIGURE 2 NOE differences between the proximal hydrogens of 10a and 10b.
of H-2ꢁ ↔ H-4ꢁ, which showed 2ꢁ,4ꢁ-cis relationships, was observed. According
to this result, 4ꢁ-vinyl and 2ꢁ-purine base of 10b were located on the β face.
On the other hand, for 10a compound, weak NOE (0.7%), such as H-2ꢁ ↔
H-4ꢁ, was assigned to the 2ꢁ,4ꢁ-trans relationships.
Cross-metathesis[12] of 10b with diethylphosphonate using 2nd-
generation Grubbs catalyst[13] gave vinylidene phosphonate nucleoside ana-
logue 11, which was reduced in transfer catalytic hydrogenation condi-
tions[14] to give phosphonate nucleoside analogue 12 in 51% two-step yield.
The chlorine group of purine analogue 12 was then converted to amine
with methanolic ammonia at 60◦C to give a corresponding adenosine phos-
phonate derivative 13. Hydrolysis of diethyl phosphonate functional groups
of 13 by treatment with bromotrimethylsilane in CH3CN in the presence
of 2,6-lutidine gave an adenosine phosphonic acid derivative 14.[15] For
the synthesis of guanine analogues, 2-fluoro-6-chloropurine[16] was con-
densed with glycosyl donor in similar conditions used for the condensa-
tion of 6-chloropurine. Vorbruggen coupling[17] of acetate 9 with 2-fluoro-
6-chloropurine gives analogues 15a and 15b. Cross-metathesis of 15b and
diethylvinylphosphonate gave 16 in 60% yield, which was catalytically re-
duced to 17 under similar procedure for 12.
Bubbling ammonia into compound 17 gave separable 2-fluoro-6-
aminopurine analogue[18] 18a (11%) and 2-amino-6-chloropurine analogue
18b (44%). 2-Amino-6-chloropurine derivative 18b was treated with TMSBr
to provide phosphonic acid and sequentially treated sodium methoxide and
2-mercaptoethanol in methanol to give desired guanine phosphonic acid 19
(Scheme 2).[19]
The antiviral activity of phosphonate nucleoside is mostly explained
by its intracellular metabolism to their diphosphates followed by incorpo-
ration into the viral genome and chain termination.[20] MT-4 cells (1 ×
105 cell/mL) were infected with HIV-1 (HTLV-IIIB strain) at a multiplicity
of infection (MOI) of 0.02 and were cultured in the presence of various
concentrations of test compounds. After a 4-day incubation at 37◦C, the
number of viable cells was monitored by the 3-(4,5-di-methylthiazole-2-yl)-
2,5-diphenyltetrazolium bromide method. The cytotoxicity of compounds

Threosyl 5 ꢁ-deoxyphosphonic Acid Purine Analogues
415
SCHEME 2 Synthesis of threosyl-5ꢁ-deoxyphosphonate guanine analogue.
was evaluated in parallel with their antiviral activity, based on the viability of
mock-infected cells.[21] The synthesized compounds 14 and 19 were tested
against HIV-1 (MT-4 cells). In particular, the adenine analogue 14 did show
moderate antiviral activity against HIV-1 (Table 1), indicating that this virus
might allow the sugar moiety for diphosphorylation or some affinity of its
diphosphate toward viral polymerases.
In summary, a novel synthetic method for threosyl 5ꢁ-deoxyphosphonic
acids purine nucleosides from 1,4-dihydroxy-2-butene was developed. When
the synthesized compounds were tested against HIV-1, the adenine analogue
14 exhibited some antiviral activity. The information obtained in the present

416
L. J. Liu et al.
TABLE 1 The antiviral activities of the synthesized compounds
Cytotoxicity CC50
HIV-1
(µg/mL)
Compound
EC50 (µg/mL)
EC90 (µg/mL)
CEM
Vero
14
19
PMEA
AZT
12.6
60.2
5.4
80
95
ND
ND
30.4
>100
50.3
>100
>100
>100
51.2
0.16
14.7
ND: Not determined.
PMEA: 9-(2-(Phosphonomethoxy)ethyl)adenine.
AZT: Azidothymidine.
study will be useful for the development of a novel nucleoside analogue. In
addition, studies toward cell permeability, with an S-acyl-2-thioethyl (SATE)
prodrug of phosphonic acid analogues, are under way.
EXPERIMENTAL SECTION
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). UV
spectra were obtained on a Beckman DU-7 spectrophotometer (Beckman,
South Pasadena, CA, USA). MS spectra were collected in electrospray ion-
ization (ESI) mode. The elemental analyses were performed using a Perkin-
Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA). TLC was performed
on Uniplates (silica gel) purchased from Analtech Co. (7558, Newark, DE,
USA). All reactions were carried out under an atmosphere of nitrogen un-
less specified. Dry dichloromethane, benzene and pyridine were obtained
by distillation from CaH2. Dry THF was obtained by distillation from Na and
benzophenone immediately prior to use.
( )-4-Vinyl-dihydrofuran-2-one (7): To a solution of 6 (1.6 g, 5.8 mmol)
in THF (10 mL), TBAF (8.8 mL, 1.0 M solution in THF) was added at 0◦C.
The mixture was stirred overnight at rt and concentrated in vacuo. The
residue was purified by silica gel column chromatography (Hexane/EtOAc,
10:1) to give 11 (450 mg, 68%):1H NMR (CDCl3, 300 MHz) δ 5.71–5.68 (m,
1H), 5.08–5.01 (m, 2H), 4.34–4.30 (m, 2H), 3.79 (m, 1H), 2.32 (dd, J = 9.8,
6.6 Hz, 1H), 2.26 (dd, J = 9.8, 8.2 Hz, 1H);13C NMR (CDCl3, 75 MHz) δ
173.2, 143.1, 112.3, 76.2, 42.6, 32.7; MS m/z 113 (M+H)+.
( )-4-Vinyl-tetrahydrofuran-2-ol (8): To a cooled (−78◦C), stirred solu-
tion of lactone 7 (250 mg, 2.23 mmol) in dry toluene (10 mL) was added

Threosyl 5 ꢁ-deoxyphosphonic Acid Purine Analogues
417
dropwise a 1.0 M solution of diisobutylaluminium hydride (DIBALH) (2.45
mL, 2.45 mmol). The reaction was stirred for 30 minutes at −78◦C, followed
by dropwise addition of methanol (2.5 mL) and diluted with 50% ethyl ac-
etate/hexane. The reaction mixture was warmed to room temperature and
stirred for 2 hours, and the precipitate was removed by filtration through
a pad of Celite, washed with ethyl acetate. The filtrate and washings were
concentrated in vacuo and the residue was purified by silica gel column chro-
matography (EtOAc/hexane, 1:8) to give 8 (221 mg, 87%) as a colorless oil:
1H NMR (CDCl3, 300 MHz) δ 5.73–5.69 (m, 1H), 5.50 (m, 1H), 5.02–4.99
(m, 2H), 3.77–3.74 (m, 2H), 2.56 (m, 1H), 2.03–2.97 (m, 2H); MS m/z 115
(M+H)+.
( )-Acetic Acid 4-Vinyl-tetrahydrofuran-2-yl Ester (9): To a solution of
compound 8 (271 mg, 2.37 mmol) in anhydrous pyridine (12 mL), Ac2O
(0.355 g, 3.5 mmol) was slowly added, and the mixture was stirred overnight
under nitrogen. The pyridine was evaporated under reduced pressure and
co-evaporated with toluene. The residue was diluted with H2O (100 mL),
extracted with EtOAc (100 mL), dried over MgSO4, and filtered. The filtrate
was concentrated under reduced pressure. The residue was purified by silica
gel column chromatography (EtOAc/hexane, 1:20) to give compound 9
(300 mg, 81%) as a colorless oil: 1H NMR (CDCl3, 300 MHz) δ 6.23–6.19 (m,
1H), 5.71–5.66 (m, 1H), 5.07–4.97 (m, 2H), 3.78–3.73 (m, 2H), 2.52–2.45
(m, 3H), 2.03 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 171.6, 143.4, 112.1,
102.0, 100.8, 67.3, 36.6, 34.2, 18.3, 17.8; MS m/z 157 (M+H)+.
(rel)-(2R,4R)-9-(4-Vinyl-tetrahydrofuran-2-yl) 6-Chloropurine (10a) and
(rel)-(2S,4R)-9-(4-Vinyl-tetrahydrofuran-2-yl) 6-Chloropurine (10b): 6-
Chloropurine (264 mg, 1.712 mmol), anhydrous HMDS (10 mL), and a
catalytic amount of ammonium sulfate (20 mg) were refluxed to a clear
solution, and the solvent was distilled under anhydrous conditions. The
residue was dissolved in anhydrous 1,2-dichloroethane (12 mL). To this
mixture, a solution of 9 (156.2 mg, 1.00 mmol) in dry DCE (10 mL) and
TMSOTf (380 mg, 1.712 mmol) was added, and the resulting mixture was
stirred at rt for 3 hours. The reaction mixture was quenched with 3 mL of
saturated NaHCO3 and stirred for 1 hour. The resulting solid was filtered
through a Celite pad, and the filtrate was extracted with CH2Cl2 twice. The
combined organic layers were dried over anhydrous MgSO4, filtered, and
concentrated under vacuum. The residue was purified by silica gel column
chromatography (EtOAc/hexane, 3:1) to give compound 10a (85 mg, 34%)
and 10b (87 mg, 35%). Data for 10a: 1H NMR (CDCl3, 300 MHz) δ 8.78 (s,
1H), 8.34 (s, 1H), 5.94 (dd, J = 6.8, 4.2 Hz, 1H), 5.78 (m, 1H), 5.10–5.02
(m, 2H), 3.76–3.69 (m, 2H), 2.57 (m, 1H), 2.31 (m, 1H), 2.24 (dd, J = 10.0,
6.8 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ 151.8, 151.5, 151.2, 144.8, 142.5,
132.7, 111.5, 87.1, 69.2, 36.7, 34.1; Anal. Calc. for C11H11ClN4O: C, 52.70;
H, 4.42; N, 22.35. Found: C, 52.68; H, 4.39; N, 22.36; MS m/z 251 (M +
H)+. Data for 10b: 1H NMR (CDCl3, 300 MHz) δ 8.75 (s, 1H), 8.31 (s, 1H),

418
L. J. Liu et al.
5.96 (dd, J = 6.0, 1.8 Hz, 1H), 5.76 (m, 1H), 5.09–5.01 (m, 2H), 3.78 (m,
1H), 3.69 (dd, J = 6.2, 4.8 Hz, 1H), 2.55 (m, 1H), 2.34 (dd, J = 9.9, 6.2 Hz,
1H), 2.24 (dd, J = 10.0, 4.6 Hz, 1H); 13C NMR (CDCl3, 75 MHz) δ 151.6,
151.4, 151.0, 144.4, 143.0, 132.4, 111.7, 88.6, 68.7, 37.2, 34.3; Anal. Calc.
for C11H11ClN4O: C, 52.70; H, 4.42; N, 22.35. Found: C, 52.72; H, 4.41; N,
22.32; MS m/z 251 (M+H)+.
(rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-vinyl-tetrahydrofuran-2-yl) 6-chloropurine}
phosphonate (11): To a CH2Cl2 (5 mL) solution of purine analogue 10b
(159 mg, 0.412 mmol) and 4-equivalent of diethyl vinylphosphonate (270
mg, 1.65 mmol), 2nd-generation Grubbs catalyst (17.52 mg, 0.0206 mmol)
was added. The reaction mixture was refluxed for 28 hours under dry argon
gas and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (EtOAc/n-Hexane/MeOH, 3:1:0.03) to
give 11 (102 mg, 64%) as a form: 1H NMR (CDCl3, 300 MHz) δ 8.71 (s, 1H),
8.27 (s, 1H), 6.53 (m, 1H), 5.96 (dd, J = 4.2, 6.2. Hz, 1H), 5.67 (d, J = 5.2
Hz, 1H), 4.16-4.11 (m, 4H), 3.79 (dd, J = 9.6, 5.2 Hz, 1H), 3.52 (dd, J = 9.6,
6.8 Hz, 1H), 2.50–2.42 (m, 2H), 2.18 (dd, J = 10.8, 8.2 Hz, 1H), 1.31 (m,
6H);13C NMR (CDCl3, 75 MHz) δ 151.7, 151.4, 153.2, 150.9, 144.6, 133.1,
110.8, 87.1, 67.9, 63.2, 36.5, 34.8, 14.6; Anal. Calc. for C15H20ClN4O4P: C,
46.58; H, 5.21; N, 14.49. Found: C, 46.62; H, 5.19; N, 14.51; MS m/z 387
(M+H)+.
(rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-ethyltetrahydrofuran-2-yl) 6-chloropurine}
phosphonate (12): A solution of vinyl phosphonate analogue 11 (839 mg,
2.17 mmol) in methanol (18 mL) was added 10% Pd/C (23 mg) and cyclo-
hexene (6.0 mL) under Ar. The reaction mixture was refluxed for 30 hours
and filtered through a pad of Celite, evaporated, and purified by silica gel
column chromatography using methanol and ethyl acetate (1:20) to give
1
ethyl phosphonate analogue 12 (683 mg, 81%) as a white solid: H NMR
(CDCl3, 300 MHz) δ 8.73 (s, 1H), 8.32 (s, 1H), 5.97 (t, J = 4.8 Hz, 1H),
4.22–4.15 (m, 6H), 3.76–3.67 (m, 2H), 2.40 (dd, J = 10.4, 5.6 Hz, 1H),
2.22 (dd, J = 10.5, 8.6 Hz, 1H), 1.81 (m, 1H), 1.33–1.30 (m, 8H);13C NMR
(CDCl3, 75 MHz) δ 151.6, 151.3, 150.8, 144.3, 132.8, 86.4, 68.7, 62.6, 61.9,
35.0, 33.4, 24.2, 16.2, 14.2; Anal. Calc. for C15H22ClN4O4P (+1.0 MeOH):
C, 45.66; H, 6.24; N, 13.31. Found: C, 45.68; H, 6.23; N, 13.29; MS m/z 389
(M+H)+.
(rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-ethyltetrahydrofuran-2-yl) adenine} phos-
phonate (13): A solution of 12 (125 mg, 0.321 mmol) in saturated methano-
lic ammonia (10 mL) was stirred overnight at 60∞C in a steel bomb, and
the volatiles were evaporated. The residue was purified by silica gel column
chromatography (MeOH/CH2Cl2, 1:12) to give 13 (70 mg, 59%) as a white
solid: mp 162–164∞C; UV (MeOH) λmax 260.5 nm; 1H NMR (DMSO-d6, 300
MHz) δ 8.34 (s, 1H), 8.18 (s, 1H), 5.96 (dd, J = 6.0, 4.2 Hz, 1H), 4.08–4.04
(m, 2H), 3.72–3.65 (m, 6H), 1.83 (m, 1H), 1.32–1.28 (m, 8H); 13C NMR
(CDCl3, 75 MHz) δ 154.7, 147.8, 152.2, 144.7, 128.8, 87.5, 67.8, 62.7, 62.0,

Threosyl 5 ꢁ-deoxyphosphonic Acid Purine Analogues
419
35.4, 33.2, 15.6, 14.2; Anal. Calc. for C15H24N5O4P (+1.0 MeOH): C, 47.87;
H, 7.03; N, 17.44. Found: C, 47.88; H, 7.01; N, 17.47; MS m/z 370 (M+H)+.
(rel)-(2ꢁR,4ꢁR)-{9-(4-Ethyl-tetrahydrofuran-2-yl) adenine} phosphonic
acid (14): To a solution of phosphonate 13 (124 mg, 0.336 mmol) in
anhydrous CH3CN (8 mL) and 2,6-lutidine (0.782 mL, 6.72 mmol) was
added trimethylsilyl bromide (0.514 mg, 3.36 mmol). The mixture was
heated overnight at 70◦C under nitrogen gas and then concentrated in
vacuo. The residue was partitioned between CH2Cl2 (70 mL) and purified
water (70 mL). The aqueous layer was washed with CH2Cl2 (2 × 60 mL) and
then freeze-dried to give phosphonic acid 14 (77.9 mg, 74%) as a yellowish
1
foam: UV (H2O) λmax 261.5 nm; H NMR (DMSO-d6, 300 MHz) δ 8.34 (s,
1H), 8.17 (s, 1H), 6.00 (dd, J = 3.8, 6.5 Hz, 1H), 4.04 (m, 2H), 3.71 (dd,
J = 8.2, 10.2 Hz, 1H), 3.54 (dd, J = 6.4, 10.1 Hz, 1H), 2.27–2.20 (m, 2H),
1.31 (m, 2H); 13C NMR (DMSO-d6, 75 MHz) δ 154.5, 152.3, 147.5, 141.8,
128.4, 87.2, 68.3, 35.2, 33.6, 27.8, 15.3; Anal. Calc. for C11H16N5O4P (+2.0
H2O): C, 37.82; H, 5.77; N, 20.05. Found: C, 37.79; H, 5.75; N, 20.02; MS
m/z 314 (M+H)+.
(rel)-(2ꢁR,4ꢁS)-Vinyl-tetrahydrofuran-2-yl) 2-fluoro-6-chloropurine (15a)
and
(rel)-(2ꢁR,4ꢁR)-Vinyl-tetrahydrofuran-2-yl)
2-fluoro-6-chloropurine
(15b): Coupling of 9 with 2-fluoro-6-chloropurine under the similar
reaction condition as described for 10 to give 15a and 15b. Data for 15a:
yield 33%; UV (MeOH) λmax 269.0 nm; 1H NMR (CDCl3, 300 MHz) δ 8.45
(s, 1H), 5.97 (t, J = 5.4 Hz, 1H), 5.72 (m, 1H), 5.05–4.95 (m, 2H), 3.76–3.71
(dd, J = 6.2, 10.8, 1H), 3.51–3.46 (dd, J = 8.4, 10.8 Hz, 1H), 2.56 (m, 1H),
2.32 (m, 1H), 2.21 (dd, J = 8.0, 10.4 Hz, 1H); 13C NMR (CDCl3, 75 MHz)
δ 154.7, 152.3, 147.8, 143.5, 137.2, 128.6, 112.5, 85.9, 67.8, 36.4, 34.5; Anal.
Calc. for C11H10ClFN4O: C, 49.17; H, 3.75; N, 20.85. Found: C, 49.15; H,
3.74; N, 20.83; MS m/z 269 (M+H)+. Data for 15b: yield 35%; UV (MeOH)
1
λmax 268.0 nm; H NMR (CDCl3, 300 MHz) δ 8.45 (s, 1H), 5.99 (dd, J =
5.8, 3.6 Hz, 1H), 5.72–5.68 (m, 1H), 5.07–4.96 (m, 2H), 3.81–3.75 (m, 2H),
2.57–2.46 (m, 2H), 2.32 (dd, J = 6.8, 10.2 Hz, 1H); 13C NMR (CDCl3, 75
MHz) δ 154.7, 152.5, 148.3, 144.3, 136.5, 128.8, 112.4, 87.0, 67.9, 36.8, 34.3;
Anal. Calc. for C11H10ClFN4O: C, 49.17; H, 3.75; N, 20.85. Found: C, 49.20;
H, 3.74; N, 20.87; MS m/z 269 (M+H)+.
(rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-vinyl-tetrahydrofuran-2-yl) 2-fluoro-6-chloro-
purine} phosphonate (16): Phosphonate nucleoside analogue 16 was pre-
pared from 15b using a similar cross metathesis procedure as described for
1
11: yield 60%; H NMR (CDCl3, 300 MHz) δ 8.49 (s, 1H), 6.62–6.57 (dd, J
= 5.8, 6.8 Hz, 1H), 5.95 (dd, J = 1.2, 5.0 Hz, 1H), 5.71 (d, J = 5.8 Hz, 1H),
4.15–4.09 (m, 4H), 3.78 (dd, J = 4.2, 9.2 Hz, 1H), 3.62 (dd, J = 6.8, 9.3
Hz, 1H), 2.55 (m, 1H), 2.34 (dd, J = 6.4, 10.4 Hz, 1H), 2.23 (dd, J = 8.2,
10.4 Hz, 1H), 1.32 (m, 6H);13C NMR (CDCl3, 75 MHz) δ 154.9, 153.1, 152.5,
146.5, 128.6, 113.4, 109.7, 87.4, 68.2, 62.4, 61.8, 37.4, 35.2, 14.7; Anal. Calc.

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for C15H19ClFN4O4P: C, 44.51; H, 4.73; N, 13.84. Found: C, 44.48; H, 4.71;
N, 13.82; MS m/z 405 (M+H)+.
(rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-ethyl-tetrahydrofuran-2-yl) 2-fluoro-6-chloro-
purine} phosphonate (17): Compound 17 was synthesized from 16 by the
similar transfer catalytic hydrogenation procedure as described for 12: yield
1
72%; H NMR (CDCl3, 300 MHz) δ 8.43 (s, 1H), 5.98 (dd, J = 2.0, 6.2
Hz, 1H), 4.21–4.16 (m, 4H), 4.09–4.03 (m, 2H), 1.73 (m, 2H), 1.31 (m,
6H);13C NMR (CDCl3, 75 MHz) δ 154.9, 152.4, 148.4, 143.2, 125.6, 87.2,
67.7, 62.0, 61.5, 35.6, 33.9, 24.2, 16.6, 14.1; Anal. Calc. for C15H21ClFN4O4P
(+1.0 MeOH): C, 43.79; H, 5.74; N, 12.76. Found: C, 43.81; H, 5.75; N, 12.75;
MS m/z 407 (M+H)+.
(rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-ethyl-tetrahydrofuran-2-yl) 2-fluoro-6-amino-
purine} phosphonate (18a) and (rel)-(2ꢁR,4ꢁR)-Diethyl {9-(4-ethyl-tetrahy-
drofuran-2-yl) 2-amino-6-chloropurine} phosphonate (18b): Dry ammonia
gas was bubbled into a stirred solution of 17 (390 mg, 0.96 mmol) in DME
(18.4 mL) at room temperature overnight. The salts were removed by filtra-
tion and the filtrate was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (MeOH/CH2Cl2, 1:8) to
give 18a (41 mg, 11%) and 18b (170 mg, 44%), respectively. Data for 18a:
UV (MeOH) λmax 261.5 nm; 1H NMR (DMSO-d6, 300 MHz) δ 8.18 (s, 1H),
7.76 (br s, NH2, 2H, D2O exchangeable), 5.98 (dd, J = 3.2, 5.8. Hz, 1H),
4.13 (m, 4H), 4.06–4.01 (m, 2H), 3.76 (dd, J = 6.8, 10.4 Hz, 1H), 3.53 (dd,
J = 8.2, 10.5 Hz, 1H), 2.36 (dd, J = 8.0, 10.8 Hz, 1H), 2.23 (dd, J = 6.2,
10.6 Hz, 1H), 1.88 (m, 1H), 1.22–1.20 (m, 6H); 13C NMR (CDCl3, 75 MHz)
δ 155.1, 152.4, 147.4, 143.5, 123.7, 87.4, 68.8, 61.9, 61.4, 35.5, 33.2, 24.6,
15.7, 13.5; Anal. Calc. for C15H23FN5O4P (+0.5 MeOH): C, 46.15; H, 6.24;
N, 17.36. Found: C, 46.17; H, 6.22; N, 17.35; MS m/z 388 (M+H)+. Data for
18b: UV (MeOH) λmax 307.5 nm; 1H NMR (DMSO-d6, 300 MHz) δ 8.12 (s,
1H), 7.68 (br s, NH2, 2H, D2O exchangeable), 5.94 (dd, J = 3.4, 5.8. Hz,
1H), 4.15–4.11 (m, 4H), 4.07–4.03 (m, 2H), 3.71–3.65 (m, 2H), 2.36 (dd, J
= 6.8, 10.8 Hz, 1H), 2.28 (dd, J = 8.6, 10.7 Hz, 1H), 1.87 (m, 1H), 1.23–1.20
(m, 6H); 13C NMR (CDCl3, 75 MHz) δ 158.3, 154.4, 151.2, 143.2, 124.6, 86.7,
67.9, 62.4, 61.9, 36.2, 33.0, 25.3, 16.2, 13.6; Anal. Calc. for C15H23ClN5O4P
(+1.0 MeOH): C, 44.09; H, 6.24; N, 16.06. Found: C, 44.11; H, 6.26; N, 16.05;
MS m/z 404 (M+H)+.
(rel)-(2ꢁR,4ꢁR)-9-{(4-Ethyl-tetrahydrofuran-2-yl) guanine} phosphonic
acid (19): To a solution of 18b (53.3 mg, 0.132 mmol) dry CH3CN (12
mL) was added trimethylsilyl bromide (0.0304 mL, 2.3 mmol) at room
temperature. After this mixture was stirred for 24 hours, the solvent was
removed, co-evaporating three times with methanol. The residue was
dissolved in MeOH (5.0 mL) and 2-mercaptoethanol (36 µL, 0.528 mmol)
and NaOMe (28 mg, 0.528 mmol) was added to the mixture. The mixture
was refluxed for 7 hours under N2, cooled, neutralized with glacial AcOH,
and evaporated to dryness under vacuum. The residue was purified by

Threosyl 5 ꢁ-deoxyphosphonic Acid Purine Analogues
421
chromatography on a column of reversed-phase C18 silica gel eluting water
1
to give 19 (27 mg, 62%) as a white solid. UV (H2O) λmax 254.5 nm; H
NMR (DMSO-d6, 300 MHz) δ 10.7 (br s, NH, H, D2O exchangeable), 8.01
(s, 1H), 6.96 (br s, NH2, 2H, D2O exchangeable), 5.97 (dd, J = 3.4, 6.2. Hz,
1H), 3.75 (dd, J = 8.2, 10.6 Hz, 1H), 3.58 (dd, J = 6.2, 10.6 Hz, 1H), 2.31
(dd, J = 6.8, 11.0 Hz, 1H), 2.22 (dd, J = 8.2, 11.0 Hz, 1H), 1.83 (m, 1H),
1.35–1.32 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 157.8, 154.2, 152.4, 136.5,
118.1, 76.3, 68.2, 36.2, 34.6, 27.8, 14.4; Anal. Calc. for C11H16N5O5P (+2.0
H2O): C, 36.17; H, 5.52; N, 19.17. Found: C, 36.18; H, 5.50; N, 19.15; MS
m/z 330 (M+H)+.
Antiviral assay. MT-4 cells (1 × 105 cell/mL) were infected with HIV-1
(HTLV-IIIB strain) at a multiplicity of infection (MOI) of 0.02 and were
cultured in the presence of various concentrations of test compounds. After
a four-day incubation at 37◦C, the number of viable cells was monitored by
the 3-(4,5-di-methylthiazole-2-yl)-2,5-diphenyltetrazolium bromide method.
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