Synthesis and anti-retroviral activity of novel 5′-deoxy-5′,5′-difluoro-threosyl nucleoside phosphonic acid analogs

Article abstract of DOI:10.1002/bkcs.10390

Novel 5′-deoxy-5′,5′-difluoro-threose purine phosphonic acid analogs were designed and synthesized from 2-propanone-1,3-diacetate. Direct displacement of the triflate intermediate 12 with diethyl (lithiodifluoromethyl) phosphonate provided the correspondi

Full text of DOI:10.1002/bkcs.10390

Article
DOI: 10.1002/bkcs.10390
BULLETIN OF THE
S. Kim et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Anti-Retroviral Activity of Novel 50-Deoxy-50,50-
difluoro-threosyl Nucleoside Phosphonic Acid Analogs
*
Seyeon Kim, Jun-Pil Jee, and Joon Hee Hong
BK-21 Project Team, College of Pharmacy, Chosun University, Kwangju 501-759, Republic of Korea.
*E-mail: hongjh@chosun.ac.kr
Received April 6, 2015, Accepted April 14, 2015, Published online July 20, 2015
Novel 50-deoxy-50,50-difluoro-threose purine phosphonic acid analogs were designed and synthesized from 2-
propanone-1,3-diacetate. Direct displacementof the triflate intermediate 12 with diethyl (lithiodifluoromethyl)
phosphonate provided the corresponding (α,α-difluoroalkyl) phosphonate 13. Condensation successfully pro-
ceeded from a glycosyl donor 14 under Vorbrüggen conditions to provide the nucleoside phosphonate analogs
15α, 15β, 18α, and 18β, respectively. Ammonolysis and hydrolysis of the phosphonates 15β and 18β yielded
the nucleoside phosphonic acid analogs 16, 17, 19b, and 20. Also, synthesis of prodrugs of adenine derivative
was performed to increase cellular uptake. The synthesized nucleoside analogs were subjected to antiviral
screening against human immunodeficiency virus (HIV)-1. Bis(SATE) prodrug 22 of the adenine analog
exhibited significant in vitro activity against HIV-1.
Keywords: Anti-human immunodeficiency virus agents, 50-Deoxy-50,50-difluoro-threosyl phosphonic acid
analog, Vorbrüggen reaction
Introduction
methoxyethylacrylate) (PMEA) analog wasprepared byChen
et al. in order to improve the biological properties of PMEA.
Among them, the monoester derivative 2 exhibited significant
activity against both human cytomegalovirus (HCMV) and
Epstein–Barr virus (EBV).8
The fluorine atom plays an important role in medicinal chem-
istry because fluorine substitution has a strong impact on the
physical, chemical, and biological properties of bioactive
compounds.1 Such fluorine modifications have been exten-
sively studied among the pharmaceutically important class
of nucleoside phosphonates, which are nucleotide analogs
in which the phosphate group is replaced by the enzymatically
and chemically stable phosphonate moiety. The fluorinated
nucleoside phosphonates abound with antiviral, antiparasitic,
and anticancer properties because they are able to act as inhi-
bitors of important enzymes of nucleoside and nucleotide
metabolism.2
Threose 50-nor nucleoside phosphonates,9 such as PMDTA
(3, EC50 = 4.5 μM) and PMDTT (4), have been assembled
from natural precursor molecules (Figure 1). Furthermore,
threose nucleic acids (TNAs) form thermally stable duplexes
with DNA and RNA that resemble the natural associations of
nucleic acids.10 Moreover, diphosphates in threose nucleo-
sides are accepted as substrates by several polymerases and
can be enzymatically incorporated into DNA.11 In addition,
Phosphonic acids often exhibit important biological proper-
ties because of their similarity to phosphates.3 The carbon–
phoshophorus bond in phosphonates, unlike phosphates, is
not susceptible to the hydrolytic action of phosphatases,
thereby imparting greater stability under physiological condi-
tions. Inparticular, alkylphosphonate esters ofnucleosidesare
generally more stable to nucleases and have greater cell per-
meability.4 It has been suggested by Blackburn and Kent5 that
α-fluoro and α,α-difluoromethylphosphonates should mimic
phosphate esters better than the corresponding phosphonates.6
This assumption was based on electronic and steric considera-
tions. Groups such as CHF and CF2 could be incorporated
either in place of 30- or 50-oxygens, or as CFH2, CF2H, or
CF3 in place of the hydroxyl on the phosphates. 9-(5,5-
difluoro-5-phosphonopentyl)guanine 1 has been utilized as
a multisubstrate analog inhibitor of purine nucleoside phos-
phorylase.7 Furthermore, the α-monofluorinated poly(2-
O
NH2
N
NH
NH2
N
N
O
P
N
N
F
HO
EtO
N
N
+
HO
NH4
O
P
O
F
F
O
1
2
O
N
NH2
H3C
O
N
NH
N
O
N
O
N
O
O
O
HO P
OH
HO P
OH
O
4 (PMDTT)
3 (PMDTA)
Figure 1. Synthesis rationale of 50-deoxy-50,50-difluoro-threose
nucleoside phosphonic acids as potent anti-HIV agents.
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Article
50-Deoxy-50,50-difluoro-threosyl Nucleoside Phosphonic Acid Analogs
KOREAN CHEMICAL SOCIETY
these nucleosides are accepted as substitutes for ribonucleo-
sides at the catalytic site of the hammerhead ribozyme,
although then the catalytic efficiency of ribozymes is signifi-
cantly reduced.12 PMDTA has a phosphonomethoxy group at
the 30-position of its furanose ring and no substituent at the 40-
position.13 This absence of a 40-hydroxymethyl group avoids
problems of steric hindrance during phosphorylation reactions
with kinases. Recently, we have designed and synthesized
several related structures to find potent antiviral agents in this
category.14
25 ꢀCfor18 h,andthendilutedwithhexane(25 mL).Thewhite
precipitate was filtered off through a plug of Celite and washed
with5% ethyl acetate/hexane. The filtratewas concentrated and
purified using silica gel column chromatography (EtOAc/hex-
1
ane, 1:10) to yield compound 8 (712 mg, 81%). H NMR
(CDCl3, 300 MHz) δ 7.23–7.17 (m, 5H), 4.65 (s, 2H), 4.47
(dd, J = 10.6, 5.0 Hz, 1H), 4.23 (dd, J = 10.6, 7.0 Hz, 1H),
3.48 (dd, J = 10.2, 6.8 Hz, 1H), 3.25 (dd, J = 10.2, 7.8 Hz,
1H), 2.36–2.29 (m, 2H), 2.15 (dd, J = 10.4, 6.6 Hz, 1H); 13C
NMR (CDCl3, 75 MHz) δ 175.7, 137.5, 128.5, 127.8, 127.3,
80.2, 76.4, 71.5, 36.6, 29.2.
Stimulated by the findings that 50-mono- and difluorinated
nucleoside analogs and threose 50-nornucleoside phosphonic
acids have excellent antiviral activities, we sought to
synthesize a novel class of nucleosides consisting of 50-
deoxy-50,50-difluoro-threose phosphonic acid analogs to find
more effective therapeutics against human immunodeficiency
virus (HIV).
(rel)-(1S/1R,3S)-3-[(Benzyloxy)methyl]-tetrahydrofuran-
1-ol(9):Asolutionofcompound8(451 mg, 2.19 mmol)intol-
uene (25 mL) was treated with 4.37 mL of 1 M DIBAL-H in
hexane at −78 ꢀC for 1 h. The reaction was quenched with 1
mL of methanol (MeOH) and warmed to room temperature
for 1 h before aqueous (aq) NaHCO3 (2 mL) and EtOAc
(50 mL) were added to the mixture. The resulting mixture
was filtered and the filtrate was concentrated to dryness and
purified using silica gel column chromatography (EtOAc/hex-
Experimental
1
General. Uncorrected meltingpointsweredeterminedusinga
Mel-temp II laboratory device (Brossard Quebeck CANADA
J4Y2Z2). Nuclear magnetic resonance (NMR) spectra were
recorded using a JEOL 300 Fourier transform spectrometer
(JEOL, Tokyo, Japan); chemical shifts are reported in parts
per million (δ) and signals as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), or dd (doublet of doublets). Ultravi-
olet (UV) spectra were obtained using a Beckman DU-7 spec-
trophotometer (Beckman, South Pasadena, CA, USA). Mass
spectra (MS) were collected in electrospray ionization (ESI)
mode. Elemental analyses were performed using a Perkin-
Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA).
Thin-layer chromatography (TLC) was performed on Uni-
plates (silica gel) purchased from Analtech Co. (7558; New-
ark, DE, USA). All reactions were performed in a nitrogen
atmosphere unless otherwise specified. Dry dichloromethane,
benzene, and pyridine were obtained by distillation from
CaH2. Dry tetrahydrofuran (THF) was obtained by distillation
from Na and benzophenone immediately prior to use.
( )-Dihydro-4-(hydroxymethyl)furan-2(3H)-one(7):To
a solution of lactone 6 (1.14 g, 10 mmol) in 50 mL of MeOH,
0.5 g of Pd/C (5% w/w) was added under H2 atmosphere; the
mixture was stirred for 6 h. After filtration of the reaction mix-
turethroughaCelite pad, thefiltrate wasconcentrated andpur-
ified using silica gel column chromatography (EtOAc/hexane,
1:15) to yield compound 7 (1.05 g, 91%). 1H NMR (CDCl3,
300 MHz) δ 4.42 (dd, J = 10.8, 4.8 Hz, 1H), 4.21 (dd, J =
10.8, 6.8 Hz, 1H), 3.69 (dd, J = 10.2, 6.6 Hz, 1H), 3.43 (dd,
J = 10.2, 8.0 Hz, 1H), 2.37 (dd, J = 9.8, 6.6 Hz, 1H),
2.20–2.11 (m, 2H); 13C NMR (CDCl3, 75 MHz) δ 175.4,
72.5, 69.4, 35.1, 32.6.
ane, 1:7) to yield compound 9 (405 mg, 89%) as an oil. H
NMR (CDCl3, 300 MHz) δ 7.22–7.17 (m, 5H), 5.52–5.48
(m, 1H), 4.65 (s, 2H), 3.86 (m, 1H), 3.57–3.47 (m, 2H),
3.24 (m, 1H), 2.14–2.03 (m, 3H); Anal. Calcd. for
C12H16O3: C, 69.21; H, 7.74. Found: C, 69.36; H, 7.66; MS
m/z 209 (M + H)+.
(rel)-(1S/1R,3S)-3-[(Benzyloxy)methyl]-tetrahydro-1-
methoxyfuran (10): To a stirred solution of lactol 9 (2.58 g,
12.4 mmol) in methanol (30 mL) was added 1% methanolic
hydrogen chloride solution (prepared by adding 85 μL acetyl
chloride to 5 mL MeOH). The reaction mixture was stirred for
30 min at room temperature under argon atmosphere, then
sodium bicarbonate (700 mg) added, and the stirring contin-
ued for further 10 min. The solids were filtered and the solvent
removed in vacuo to give the residue as an oil. The residue was
purified using silica gel column chromatography (EtOAc/hex-
ane, 1:20) to yield compound 10 (2.28 g, 83%) as an oil. 1H
NMR (CDCl3, 300 MHz) δ 7.21–7.16 (m, 5H), 5.13–5.09
(m, 1H), 4.62 (s, 2H), 3.87–3.85 (m, 1H), 3.54–3.50 (m,
1H), 3.44–3.42 (m, 1H), 3.21–3.18 (m, 1H), 2.21–2.18 (m,
4H), 2.09–2.01 (m, 2H); Anal. Calcd. for C13H18O3: C,
70.24; H, 8.16. Found: C, 70.11; H, 8.27; MS m/z 223 (M
+ H)+.
(rel)-(1S/1R,3S)-(Tetrahydro-1-methoxyfuran-3-yl)
methanol (11): Anhydrous ammonia (15 mL) was condensed
into a three-necked, round-bottomed flask containing a solu-
tion of methyl glycoside 10 (222 mg, 1.0 mmol) in dry tetra-
hydrofuran (3 mL) at −78 ꢀC. To this mixture was added a
minimum amount of lithium (~60 mg) sufficient to maintain
a blue color, and the resulting deep blue solution was stirred
for 30 min at −78 ꢀC for 2 min. Methanol was added dropwise
at the same temperature until the deep blue color disappeared.
The colorless solution was stirred for 30 min at −78 ꢀC, and
then solid ammonia chloride (6 g) was added. After stirring
for 1 h at −78 ꢀC, ammonia was allowed to evaporate (6 h).
Ether (40 mL) was added, and the mixture was dried over
( )-4-[(Benzyloxy)methyl]-dihydrofuran-2(3H)-one (8):
To a stirred solution of alcohol 7 (495 mg, 4.27 mmol) and ben-
zyl trichloroacetimidate in 33% methylene chloride/cyclohex-
ane (25 mL) at room temperature was added trifluoromethane
sulfonic acid (0.05 mL). The reaction mixture was stirred at
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50-Deoxy-50,50-difluoro-threosyl Nucleoside Phosphonic Acid Analogs
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anhydrous magnesium sulfate, filtered through a pad of Celite,
and washed with ether (150 mL). The filtrate and washings
were concentrated in vacuo, and the residue was purified using
silica gel column chromatography (EtOAc/hexane, 1:4) to
yield compound 11 (2.28 g, 78%) as an oil. 1H NMR
(CDCl3, 300 MHz) δ 5.09–5.05 (m, 1H), 3.83–3.79 (m,
1H), 3.61–3.55 (m, 2H), 3.39–3.34 (m, 1H), 3.26 (s, s, 3H),
2.09–1.87 (m, 3H); Anal. Calcd. for C6H12O3: C, 54.53; H,
9.15. Found: C, 54.68; H, 9.06; MS m/z 133 (M + H)+.
(rel)-(1S/1R,3R)-(Tetrahydro-1-methoxyfuran-3-yl)
methyl trifluoromethanesulfonate (12): To a cooled solu-
tion of glycoside 11 (304 mg, 2.304 mmol) in pyridine
(0.72 mL, 8.9 mmol) and CH2Cl2 (48 mL), triflic anhydride
(0.456 mL, 2.76 mmol) was slowly added. After 1.5 h, the
reaction mixture was poured onto a mixture of ice and sodium
hydrogen carbonate. The aqueous layer was extracted with
CH2Cl2 (3 × 60 mL), and the combined CH2Cl2 solution
was dried and rapidly and repeatedly concentrated with tolu-
enetoremoveanyresidual pyridine. Theresiduewasextracted
with light petroleum (3 × 60 mL), and the combined extracts
were filtered and cooled. After careful evaporation of addi-
tional solvent, the crude residue 12 (605 mg, ~100%) was sub-
jected to the next reaction without further purification.
(1S/1R,3S)-(Diethyl 5,5-difluoro-4-tetrahydro-1-meth-
oxyfuran-3-yl) ethylphosphonate (13): To a solution of dii-
sopropylamine (182 μL, 1.3 mmol) and HMPA (226 μL, 1.3
mmol) at −78 ꢀC in THF (2 mL) under Ar was added n-
butyllithium (814 μLofa1.6Msolutioninhexane, 1.3 mmol).
The resulting solution was allowed to stir for 30 min at 0 ꢀC
and then cooled to −78 ꢀC. To this solution of LDA at −78 ꢀ
C were added via a cannula, a (−78 ꢀC) solution of diethyl
(α,α-difluoromethyl) phosphonate (204 μL, 1.3 mmol) in
THF (1.0 mL), and, 2 min later, a (−78 ꢀC) solution of triflate
12 (98 mg, 0.372 mmol) inTHF (2.0 mL), dropwise, viaa can-
nula. After 10 min at −78 ꢀC, the reaction was quenched by
adding aqueous NH4Cl (6.0 mL) and Et2O (6.0 mL). The
aqueous layer was further extracted with EtOAc (2 × 20
mL), and the combined organic extracts were dried, filtered,
and evaporated. Silica gel flash chromatography (EtOAc/hex-
ane, 1:1) gave 13 (78 mg, 70%). 1H NMR (CDCl3, 300 MHz)
δ 5.12–5.09 (m, 1H), 4.34–4.26 (m, 4H), 3.83–3.79 (m, 1H),
3.55–3.51 (m, 1H), 3.23 (s, s, 3H), 2.13–1.99 (m, 5H),
1.38–1.33 (m, 6H); 31P (81 MHz, CDCl3) δ 6.67 (app t,
JP,F = 108 Hz); Anal. Calcd. for C11H21F2O5P: C, 43.71; H,
7.00. Found: C, 43.62; H, 6.96; MS m/z 303 (M + H)+.
(rel)-Diethyl 4-[(1S/1R,3S)-1-acetoxy-tetrahydrofuran-
3-yl]-5,5-difluoroethylphosphonate (14): Glycoside 13
(725 mg, 2.4 mmol) was dissolved in EtOAc (20 mL). The
solution was cooled to −15 ꢀC and mixed with a cooled
(−15 ꢀC) solution of EtOAc (40 mL), acetic anhydride (22.0
mL), acetic acid (16.6 mL) and conc. H2SO4 (0.1 mL). The
solution mixture was stirred for 18 h at 0 ꢀC. The reaction
was diluted with CHCl3 (150 mL) and poured into cold 5%
aqueous NaHCO3 (200 mL). The organic layer was separated,
and the aqueous layer was extracted with CHCl3 (3 × 50 mL).
The combined organic layers were washed with brine, dried,
and evaporated to dryness. The residue was purified using sil-
ica gel column chromatography (EtOAc/hexane, 1:2) to yield
compound 14 (641 mg, 81%) as a syrup. H NMR (CDCl3,
300 MHz) δ 6.23–6.19 (m, 1H), 4.32–4.24 (app quintet, J =
7.0 Hz, 4H), 3.86–3.81 (m, 1H), 3.61–3.56 (m, 1H),
2.24–2.19 (m, 4H), 2.04 (s, s, 3H), 1.86 (m, 1H), 1.36–1.32
(m, 6H); 31P (81 MHz, CDCl3) δ 6.87 (t, JP,F = 98 Hz); Anal.
Calcd. for C12H21F2O6P: C, 43.64; H, 6.41. Found: C,
43.79; H, 6.36; MS m/z 331 (M + H)+.
1
(rel)-Diethyl 4-[(1S,3S)-1-(6-chloro-9H-purin-9-yl)-tet-
rahydrofuran-3-yl]-5,5-difluoroethylphosphonate (15α)
and (rel)-diethyl 4-[(1R,3S)-1-(6-chloro-9H-purin-9-yl)-
tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate
(15β): 6-Chloropurine (260 mg, 1.68 mmol), anhydrous
HMDS (12 mL), and a catalytic amount of ammonium sulfate
(16.8 mg) were refluxed to a clear solution (10 h); the solvent
was then distilled under anhydrous conditions. The residue
obtained was dissolved in anhydrous 1,2-dichloroethane
(10 mL), and to this mixture a solution of 14 (277 mg, 0.84
mmol) in dry dichloroethane (DCE) (12 mL) and TMSOTf
(373 mg, 1.68 mmol) was added, and stirred for 4 h at room
temperature. The reaction mixture was quenched with 6.0
mL of saturated NaHCO3, stirred for 2 h, filtered through a
Celite pad, and the filtrate obtained was then extracted twice
with CH2Cl2 (2 × 100 mL). The combined organic layers were
driedover anhydrous MgSO4, filtered, andconcentratedunder
vacuum. The residue was purified using silica gel column
chromatography (EtOAc/hexane/MeOH, 3:1:0.03) to yield
compounds 15α (114 mg, 32%) and 15β (117 mg, 33%). Data
for 15α: 1H NMR (CDCl3, 300 MHz) δ 8.71 (s, 1H), 8.29 (s,
1H), 6.03 (dd, J = 5.8, 4.0 Hz, 1H), 4.23–4.18 (app quintet,
J = 7.2 Hz, 4H), 3.81 (dd, J = 10.6, 8.0 Hz, 1H), 3.67 (dd,
J = 10.6, 6.4 Hz, 1H), 2.17–2.10 (m, 2H), 2.05–1.95 (m,
3H), 1.35–1.30 (app t, J = 7.2 Hz, 6H); 31P (81 MHz, CDCl3)
δ
6.89
(t,
JP,F = 99.2 Hz);
Anal.
Calc.
for
C15H20ClF2N4O4P: C, 42.41; H, 4.75; N, 13.19. Found: C,
42.38; H, 4.86; N, 13.22; MS m/z 425 (M + H)+; Data for
15β: H NMR (CDCl3, 300 MHz) δ 8.74 (s, 1H), 8.32 (s,
1
1H), 5.99 (t, J = 6.0 Hz, 1H), 4.25–4.20 (app quintet, J =
7.1 Hz, 4H), 3.85 (dd, J = 10.2, 7.6 Hz, 1H), 3.63 (dd, J =
10.3, 6.6 Hz, 1H), 2.21–2.15 (m, 2H), 2.09–1.98 (m, 3H),
1.34–1.28 (app t, J = 7.2 Hz, 6H); 31P (81 MHz, CDCl3) δ
6.85
(t,
JP,F = 99.4 Hz);
Anal.
Calcd.
for
C15H20ClF2N4O4P: C, 42.41; H, 4.75; N, 13.19. Found: C,
42.56; H, 4.61; N, 13.13; MS m/z 425 (M + H)+.
(rel)-Diethyl 4-[(1R,3S)-1-(6-amino-9H-purin-9-yl)-tet-
rahydrofuran-3-yl]-5,5-difluoroethyl phosphonate (16):
A solution of 15β (315 mg, 0.742 mmol) in saturated metha-
nolic ammonia (15 mL) was stirred overnight at 66 ꢀC in a
steel bomb and the volatiles were evaporated. The residue
was purified using silica gel column chromatography
(MeOH/CH2Cl2, 1:10) to yield 16 (186 mg, 62%) as a white
1
solid: UV (MeOH) λmax 262.0 nm; H NMR (DMSO-d6,
300 MHz) δ 8.40 (s, 1H), 8.21 (s, 1H), 5.97 (dd, J = 10.8,
8.2 Hz, 1H), 4.17 (quintet, J = 7.0 Hz, 4H), 3.86 (dd, J =
10.2, 7.2 Hz, 1H), 3.58 (dd, J = 10.2, 6.4 Hz, 1H), 2.42 (m,
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50-Deoxy-50,50-difluoro-threosyl Nucleoside Phosphonic Acid Analogs
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1H), 2.17–1.97(m, 4H), 1.34 (t, J = 7.0 Hz, 6H); 31P (81 MHz,
DMSO-d6) δ 6.79 (app t, JP,F = 102.4 Hz); Anal. Calcd. for
C15H22F2N5O4P (+1.0 MeOH): C, 43.95; H, 5.99; N, 16.02;
Found: C, 43.85; H, 6.04; N, 16.13; MS m/z 406 (M + H)+.
(rel)-4-[(1R,3S)-1-(6-Amino-9H-purin-9-yl)-tetrahy-
drofuran-3-yl]-5,5-difluoroethyl phosphonic acid (17): To
a solution of phosphonate 16 (172 mg, 0.426 mmol) in anhy-
drousCH3CN(8.5 mL)and2,6-lutidine (912 mg, 8.52 mmol),
trimethylsilyl bromide (652 mg, 4.26 mmol) was added. The
mixture was heated overnight at 75 ꢀC under nitrogen and then
concentrated in vacuo to yield a brown residue, and then co-
evaporated from conc. aq. NH4OH (2 × 21.3 mL). The result-
ant compound was purified by twice triturating the residue in
acetone (8.5 mL) and removing the acetone by evaporation.
The residue was then purified using preparative reverse-phase
chromatography. Lyophilization of the appropriate fraction
yielded the phosphonic acid salt 17 (92 mg, 59%) as a white
filtration and the filtrate was concentrated under reduced
pressure. The residue obtained was purified using silica gel
column chromatography (MeOH/CH2Cl2, 1:10) to produce
19a (29 mg, 11%) and 19b (98 mg, 39%). Data for 19a;
UV (MeOH) λmax 262.5 nm; 1H NMR (DMSO-d6, 300
MHz) δ 8.25 (s, 1H), 7.71 (br s, NH2, 2H, D2O exchangea-
ble), 5.91 (dd, J = 10.4, 7.6 Hz, 1H), 4.20 (app quintete, J
= 7.0 Hz, 4H), 3.85 (dd, J = 10.1, 6.8 Hz, 1H), 3.62 (dd, J
= 10.2, 7.8 Hz, 1H), 2.43–2.39 (m, 1H), 2.11–1.92 (m,
4H), 1.37 (app t, J = 7.0 Hz, 1H); 31P (81 MHz, DMSO-d6)
δ 6.83 (t, JP,F = 101.8 Hz); Anal. Calcd. for C15H21F3N5O4P
(+0.5 MeOH): C, 42.39; H, 5.28; N, 15.94; Found: C,
42.42; H, 5.15; N, 15.84; MS m/z 424 (M + H)+. Data for
1
19b; UV (MeOH) λmax 308.0 nm; H NMR (DMSO-d6,
300 MHz) δ 10.8 (br s, 1H, D2O exchangeable), 8.35 (s,
1H), 7.67 (br s, NH2, 2H, D2O exchangeable), 5.94 (dd, J
= 10.2, 7.4 Hz, 1H), 4.21 (app quintet, 4H), 3.85 (dd, J =
10.6, 8.0 Hz, 1H), 3.62 (dd, J = 10.6, 6.2 Hz, 1H),
2.84–2.78 (m, 1H), 2.12–1.94 (m, 4H), 1.36 (app t, 6H);
31P (81 MHz, DMSO-d6) δ 6.81 (t, JP,F = 102.8 Hz); Anal.
Calcd. for C15H21ClF2N5O4P (+1.0 MeOH): C, 40.78; H,
5.34; N, 14.86; Found: C, 40.83 H, 5.48; N, 14.91; MS m/z
440 (M + H)+.
1
salt (ammonium salt): UV (H2O) λmax 261.0 nm; H NMR
(D2O, 300 MHz) δ 8.38 (s, 1H), 8.18 (s, 1H), 5.94 (dd, J =
10.4, 7.2 Hz, 1H), 3.86 (dd, J = 10.0, 6.2 Hz, 1H), 3.62 (dd,
J = 10.0, 7.8 Hz, 2H), 2.40 (m, 1H), 2.13–1.92 (m, 4H); 13C
NMR (D2O, 75 MHz) δ 155.6, 152.7, 150.7, 141.2, 120.6,
119.2 (dt, JC,P = 204.8 Hz, JC,F = 258.2 Hz), 90.1 72.9, 36.2,
24.2 (dt, JC,P = 18.6 Hz, JC,F = 23.8 Hz), 20.4; 31P (81 MHz,
D2O) δ6.83(app t, JP,F = 104.2 Hz); HPLC tR = 10.38;HRMS
[M – H]+ req. 348.0682, found 328.0683.
(rel)-4-[(1R,3S)-1-(2-Amino-1,6-dihydro-6-oxopurin-9-
yl)-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonic
acid (20): To a solution of 19b (202 mg, 0.46 mmol) in dry
CH3CN (18.4 mL), trimethylsilyl bromide (1.40 g, 9.2 mmol)
was added at room temperature. The mixture was stirred for
20 h and the solvent was removed using co-evaporation with
MeOH thrice. The residue was dissolved in MeOH (18.4 mL)
and 2-mercaptoethanol (143 mg, 1.84 mmol), and then
NaOMe (99 mg, 1.84 mmol) was added. The mixture was
refluxed for 14 h under N2, cooled, neutralized with glacial
AcOH, and evaporated to dryness under vacuum. The residue
obtained was co-evaporated from conc. NH4OH (2 × 18.4
mL) and the resultant solid was triturated with acetone (2 ×
10.5 mL). After evaporating the acetone, the residue was pur-
ified using preparative column chromatography with reverse-
phase C18 silica gel and elution with water. Lyophilization of
the appropriate fraction yielded 20 (103 mg, 59%) as a yellow-
ish salt (ammonium salt). UV (H2O) λmax 253.0 nm; 1H NMR
(D2O, 300 MHz) δ 7.79 (s, 1H), 5.93 (dd, J = 10.4, 7.2 Hz,
1H), 3.85 (dd, J = 10.0, 7.6 Hz, 1H), 3.58 (dd, J = 10.0, 8.8
Hz, 1H), 2.42–2.37 (m, 1H), 2.15–1.97 (m, 4H); 13C NMR
(D2O, 75 MHz) δ 157.5, 154.5, 152.2, 136.4, 120.2 (dt,
JC,P = 214.0 Hz, JC,F = 258.4 Hz), 80.3, 73.7, 36.9, 25.5 (dt,
JC,P = 19.1 Hz, JC,F = 23.6 Hz), 21.5; 31P (81 MHz, D2O) δ
6.45 (app t, JP,F = 101.7 Hz); HPLC tR = 9.82 min; HRMS
[M – H]+ req. 438.0823, found 438.0824.
(rel)-Diethyl
4-[(1S,3S)-3-(6-chloro-2-fluoro-
9H-purin-9-yl)-tetrahydrofuran-1-yl]-5,5-difluoroethyl-
phosphonate (18α) and (rel)-diethyl 4-[(1R,3S)-1-(6-
chloro-2-fluoro-9H-purin-9-yl)-tetrahydrofuran-3-yl]-
5,5-difluoroethylphosphonate (18β): Condensation of 14
with 2-fluoro-6-chloropurine under Vorbrüggen condensation
conditions similar to those described for 15α and 15β yielded
18α and 18β. Data for 18α: yield 34%; UV (MeOH) λmax
1
267.5 nm; H NMR (CDCl3, 300 MHz) δ 8.51 (s, 1H), 6.02
(dd, J = 10.6, 6.8 Hz, 1H), 4.19 (app quintet, J = 7.0 Hz, 1H),
3.83 (dd, J = 9.9, 5.4 Hz, 1H), 3.59 (dd, J = 10.0, 8.2 Hz,
1H), 2.39 (m, 1H), 2.16–1.96 (m, 4H), 1.32 (app t, J = 7.0
Hz,6H);31P(81 MHz, CDCl3)δ6.87(t, JP,F = 102.0 Hz);Anal.
Calcd. for C15H19ClF3N4O4P: C, 40.69; H, 4.33; N, 12.65;
Found: C, 40.82; H, 4.41; N, 12.52; MS m/z 443 (M + H)+. data
1
for 18β: yield 35%; UV (MeOH) λmax 268.0 nm; H NMR
(CDCl3, 300 MHz) δ 8.46 (s, 1H), 5.96 (dd, J = 10.4, 8.0 Hz,
1H), 4.23 (app quintet, J = 7.0 Hz, 1H), 3.85 (dd, J = 9.8, 6.4
Hz, 1H), 3.53 (dd, J = 9.8, 7.2 Hz, 1H), 2.35–2.32 (m, 1H),
2.11–1.94 (m, 4H), 1.35 (app t, J = 7.0 Hz, 6H); 31P (81
MHz, CDCl3) δ 6.86 (t, JP,F = 102.2 Hz); Anal. Calcd. for
C15H19ClF3N4O4P: C, 40.69; H, 4.33; N, 12.65; Found: C,
40.54; H, 4.20; N, 12.76; MS m/z 443 (M + H)+.
(rel)-Diethyl 4-[(1R,3S)-1-(6-amino-2-fluoro-9H-purin-
9-yl)-tetrahydrofuran-3-yl]-5,5-difluoroethylphosphonate
(19a) and (rel)-diethyl 4-[(1R,3S)-1-(2-amino-6-chloro-9H-
purin-9-yl)-tetrahydrofuran-3-yl]-5,5-difluoroethylpho-
sphonate (19b): Dry ammonia gas was bubbled into a stirred
solution of 18β (310 mg, 0.7 mmol) in DME (15.0 mL) at
room temperature overnight. Salts were removed by
(rel)-[(1R,3S)-Bis(SATE) ester of-[1-(6-amino-9H-
purin-9-yl)-tetrahydrofuran-3-yl]-5,5-difluoroethyl phos-
phonate (22): A solution of adenine phosphonic acid deriva-
tive 17 (236 mg, 0.676 mmol) and tri-n-butylamine (378 mg,
2.04 mmol) in methanol (15 mL) was mixed for 30 min and
evaporated to dryness under reduced pressure. The residue
was thoroughly dried with anhydrous ethanol and toluene.
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50-Deoxy-50,50-difluoro-threosyl Nucleoside Phosphonic Acid Analogs
KOREAN CHEMICAL SOCIETY
The resulting foamy solid was dissolved in anhydrous pyri-
dine (40 mL) to which thioester 21 (2.08 g, 12.8 mmol) and
1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (804 mg,
2.712 mmol) were added. The mixture was stirred overnight
at room temperature and quenched with tetrabutylammonium
bicarbonate buffer (40.0 mL, 1 M solution, pH 8.0). The mix-
ture was concentrated under reduced pressure, and the residue
was diluted with water (200 mL) and extracted with CHCl3
(200 mL) two times. The combined organic layer was washed
with brine, dried over Na2SO4, filtered, and evaporated to dry-
ness under vacuum. The residue was purified by silica gel col-
umn chromatography (MeOH/Hexane/EtOAc, 0.02:3:1) to
give 22 (68 mg, 33%) as a yellow solid: m.p. 144–146 ꢀC;
UV (MeOH) λmax 262.0 nm; 1H NMR (CDCl3, 300 MHz) δ
8.31 (s, 1H), 8.15 (s, 1H), 5.99 (dd, J = 10.4, 7.2 Hz, 1H),
4.12–4.07 (m, 4H), 3.86 (dd, J = 10.3, 6.4 Hz, 1H), 3.61
(dd, J = 10.2, 7.6 Hz, 1H), 3.18–3.15 (m, 4H), 2.40 (m, 1H),
2.14–1.06 (m, 22); 13C NMR (CDCl3, 75 MHz) δ 202.7,
dissolving metal reduction for a prolonged time (~30 min) fur-
nished alcohol 11 with 78% yield, which was converted to 13
using triflation followed by a triflate displacement according
to the procedure of Berkowitz et al.16 Finally, the preparation
of a suitable glycosylating agent was achieved by acetolysis of
13 under mild acidic conditions (Ac2O, AcOH, H2SO4,
EtOAc, 0 ꢀC) to afford an anomeric mixture of 1-O-acetyl-
furanoside 14 in high yield (Scheme 2).17
The synthesis of adenine nucleoside was performed using a
Vorbrüggen condensation18 of compound 14 with silylated 6-
chloropurineandtrimethylsilyltriflate (TMSOTf )asacatalyst
in DCE to yield the protected 6-chloropurine derivatives 15α
and 15β, respectively.
A complete nuclear Overhauser effect (NOE) study led to
the unambiguous determinations of their relative stereochem-
istry (Figure 2). Compound 15β showed a strong NOE (1.2%)
of H-10 $ H-30, which indicated a 10,30-cis relationship.
According to this result, the 30-difluoromethyl phosphonate
group and the 10-purine base of 15β were located on the β face.
On the other hand, compound 15α showed a weak NOE
(0.5%) of H-10 $ H-30, indicating a 10,30-trans relationship.
The chlorine group from the purine analog 15β was then
converted to an amine with methanolic ammonia at 63 ꢀC to
produce the corresponding adenosine phosphonate derivative
16 with yield 62%. Hydrolysis of the diethyl phosphonate
functional groups of 16 with bromotrimethylsilane treatments
in CH3CN in the presence of 2,6-lutidine yielded the adeno-
sine phosphonic acid compound 17 (Scheme 3).19 For the
154.6, 152.1, 147.3, 144.3, 129.7, 119.4, 118.2 (dt, JC,P
=
211.6 Hz, JC,F = 256.2 Hz), 90.2, 71.7, 63.8 (d, J = 6.0 Hz),
49.3, 35.8, 34.9, 27.1, 25.5 (dt, JC,P = 19.1 Hz, JC,F = 21.8
Hz), 20.2; 31P (81 MHz, CDCl3) δ 6.51 (app t, JP,F = 100.8
Hz); Anal. Calcd. for C25H38F2N5O6PS2 (+1.0 MeOH): C,
46.66; H, 6.32; N, 10.96; Found: C, 46.78; H, 6.35; N,
10.88; MS m/z 638 (M + H)+.
Results and Discussion
As depicted in Scheme 1, target compound was synthesized
from butenolide 6, which was readily obtained from 2-propa-
none-1,3-diacetate 5, as previously described.15 Catalytic
hydrogenation of butenolide 6 to lactone 7 was achieved with
5% Pd/C under H2 treatment with a yield of 91%. Treatment of
7 with benzyl trichloroacetimidate and a catalytic amount of
CF3SO3H in cyclohexane/methylene chloride (2/1) at 25 ꢀC
furnished the desired O-benzyl ether 8, which was converted
to lactol 9 by DIBALH reduction in toluene at −78 ꢀC for 1.5 h
(72% yield for two steps). Protection of the anomeric position
was needed prior to phosphonation. Hence, methoxylation of
the anomeric position furnished glycoside 10 in 83% yield.
Removal of the benzyl group of glycoside 10 under routine
O
O
OMe
ii
OMe
i
HO
TfO
10
quant
91%
11
12
70%
iii
O
O
O
1
4
O
iv
81%
OMe
OAc
5
EtO P
EtO
EtO P
EtO
3
2
F
F
F
14
F
13
Reagents: i) Li, NH3, -78 oC; ii) Tf2O, pyridine, CH2Cl2; iii)
LiCF2P(O)(OEt)2, HMPA, THF; iv) Ac2O, AcOH, H2SO4, EtOAc.
Scheme 2. Synthesis of fluorinated apiose glycosyl donor intermedi-
ate 14.
O
O
O
AcO
AcO
ref.14
O
i
O
91%
HO
7
HO
iv
Cl
6
5
H
ii 81%
N
N
O
EtO P
EtO
0.5%
N
N
O
1'
O1
2'
N
O
EtO P
EtO
O
3'
O
O
O
iii
89%
BnO
N
N
OH
F
F
OMe
2
3
1
4
1
H
83%
3
3
H
N
2
2
BnO
BnO
1.2%
F F
8
9
H
10
Cl
15α
15β
Reagents: i) H2, Pd/C, EtOAc; ii) PhCH2OC(=NH)CCl3, CF3SO3H, CH2Cl2;
iii) DIBALH, toluene, -78 oC; iv) MeOH, AcCl.
Figure 2. NOE differences between the proximal hydrogens of 15α
and 16β.
Scheme 1. Synthesis of threose methyl glycoside 10.
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BULLETIN OF THE
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50-Deoxy-50,50-difluoro-threosyl Nucleoside Phosphonic Acid Analogs
KOREAN CHEMICAL SOCIETY
1
structural elucidation of 17, H, 13C, 31P NMR, and high-
First, the synthesized nucleoside phosphonate and phos-
phonic acids 16, 17, and 20 were evaluated for antiviral activ-
ity against HIV. The procedures for measuring the antiviral
activity toward wild-type HIV and cytotoxicity have been
reported previously.23 As shown in Table 1, the adenine nucle-
oside analogs 16 and 17 exhibited weak anti-HIV activity.
Furthermore, the guanine nucleoside analog 20 did not show
anti-HIV activity or cytotoxicity at concentrations up to 100 μ
M. The reason for the weak antiviral activity of the final com-
pounds might be the result of poor cell membrane diffusion of
phosphonate ions in vitro. To increase the cellular uptake of
adenine phosphonic acid analog 17, development of bis-
SATE phosphonodiester prodrugs was performed.
To synthesize the thioester prodrug of the adenine analog,
the derivative 17 was reacted with thioester 2124 in the pres-
ence of 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole
(MSNT)25 to provide the bis(SATE) derivative as a target
compound 22 (Scheme 5). Much increased antiviral anti-
HIV activity of bis(SATE) analog 22 was observed, which
was almost equivalent to that of PMDTA.
resolution mass spectrometry (HRMS) were used.
Condensation of 2-fluoro-6-chloropurine20 with glycosyl
donor 14 proceeded under conditions similar to those used
for synthesis of analogs 15α and 15β from 6-chloropurine
to yield 18α (34%) and 18β (35%), respectively.
A complete NOE study led to the unambiguous determination
of the relative stereochemistry of purine analogs 18α and 18β.
Bubbling ammonia into compound 18β yielded the 2-
fluoro-6-aminopurine21 analog 19a (11%) and the 2-amino-
6-chloropurine analog 19b (39%). The 2-amino-6-
chloropurine derivative 19b was treated with TMSBr to yield
phosphonic acid and was then treated with sodium methoxide
and 2-mercaptoethanol in methanol to produce guanosine
phosphonic acid 20 (Scheme 4).22
Cl
Cl
N
N
N
N
N
and
i
N
14
N
O
P
N
O
O
P
O
4'
EtO
Conclusion
1'
2'
5'
EtO
EtO
F
EtO
F
3'
15β (33%)
F
F
Based on the potent anti-HIV activities of the 50,50-difluoro-
nucleosides and threosyl 50-nornucleoside phosphonic acid
analogs, we designed and successfully synthesized novel 50-
deoxy-50,50-difluoro-threosyl nucleoside phosphonic acid
15α (32%)
62%
ii
NH2
N
NH2
N
N
N
N
N
iii
59%
EtO
N
O
P
N
O
O
P
O
17
Table 1. Median effective (EC50) and cytotoxic (CC50)
concentrations of the synthesized nucleoside analogs.
HO
EtO
HO
F
F
16
F
F
Compound
no.
Anti-HIV-1
Cytotoxicity
EC50 (μM)a
CC50 (μM)b
Reagents: i) Silylated 6-chloropurine, TMSOTf, DCE; ii)
NH3/MeOH; iii) TMSBr, 2,6-lutidine, CH3CN.
16
17
20
22
AZTa
PMEAb
28.2
33.4
78.6
5.7
0.002
1.56
98
Scheme 3. Synthesis of 50-deoxy-50,50-difluoro-threosyl phosphonic
>100
>100
90
>100
>100
acid adenosine analog.
Cl
N
Cl
N
N
N
N
N
N
N
and
AZT, azidothymidine; PMEA, 9-[2-(phosphonomethoxy)ethyl]adenine.
a EC50 (μM): EC50 values are for 50% inhibition of virus production as
indicated by supernatant RT levels.
i
F
F
14
O
P
O
O
O
EtO
P
EtO
b CC50 (μM): CC50 values indicate 50% cytotoxic concentration.
EtO
EtO
F
F
18α (34%)
F
F
18β (35%)
ii
X
NH2
O
N
N
NH2
N
N
N
N
N
N
N
i
NH
N
N
iii
53%
N
N
X
O
33%
O
N
O
O
P
NH2
O
N
O
P
S
P
EtO
O
O
P
O
O
HO
EtO
O
HO
O
F
F
O
F
F
HO
OH
HO
S
F
F
19a: X = NH2, Y = F (11%)
19b: X = Cl, Y = NH2 39%)
S
20
F
17
F
21
22
Reagents: i) Silylated 2-fluoro-6-chloropurine, TMSOTf, DCE; ii) NH3, DME,
rt; iii) (a) TMSBr, 2,6-lutidine, CH3CN; (b) NaOMe, HSCH2CH2OH, MeOH.
Reagents: i) thioester, 21, 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole,
pyridine.
Scheme 4. Synthesis of 50-deoxy-50,50-difluoro-threosyl phosphonic
acid guanosine analog.
Scheme 5. Synthesis of target bis(SATE) prodrug of adenine ana-
log 22.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2020–2026
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50-Deoxy-50,50-difluoro-threosyl Nucleoside Phosphonic Acid Analogs
KOREAN CHEMICAL SOCIETY
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analogs from 2-propanone-1,3-diacetate. The bis(SATE) pro-
drug analog 22 showed significant activity in a cell-based
assay compared to the phosphonic acid analogs 16, 17, and 20.
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© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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