Synthesis and biological evaluation of 1',2'-seconucleo-5'-phosphonates

Article abstract of DOI:10.1021/jm9506783

A series of 1',2'-seconucleophosphonate analogues were prepared containing adenine, cytosine, thymine, and uracil as the nucleobase. The synthetic methodology is efficient and uses chloromethyl ethers derived from the chirons diethyl (3S)-(benzyloxy)-(2R)

Full text of DOI:10.1021/jm9506783

1130
J . Med. Chem. 1996, 39, 1130-1135
Syn th esis a n d Biologica l Eva lu a tion of 1′,2′-Secon u cleo-5′-p h osp h on a tes
Saibaba Racha, Chandra Vargeese, Purushotham Vemishetti, Hussein I. El-Subbagh, Elie Abushanab,* and
Raymond P. Panzica*
Departments of Medicinal Chemistry and Chemistry, University of Rhode Island, Kingston, Rhode Island 02881
Received September 14, 1995X
A series of 1′,2′-seconucleophosphonate analogues were prepared containing adenine, cytosine,
thymine, and uracil as the nucleobase. The synthetic methodology is efficient and uses
chloromethyl ethers derived from the chirons diethyl (3S)-(benzyloxy)-(2R)-hydroxybutane-
phosphonate (1) and diethyl (3S),4-bis(benzyloxy)-(2R)-hydroxybutanephosphonate (2). Selected
deblocked derivatives, i.e., two monoesters (13 and 14), four phosphonic acids (15-18), and
one cyclic phosphonate (23), were screened for in vitro activity against certain RNA, adeno,
and HIV viruses. All of them were found to be devoid of activity.
There is considerable interest in phosphonates as
biologically active surrogates of naturally occurring
phosphates.1 Phosphonate replacement is attractive
since the carbon-phosphorus bond in phosphonates is
not susceptible to enzymatic degradation by phos-
phatases, thus enhancing physiological stability. In
addition, phosphonate esters are less polar, and this
feature gives rise to better cell permeability.2
attached the phosphonate at a late stage in the synthe-
sis8 to individual functionalized 1′,2′-seconucleosides.
The chiral phosphonate synthons have now been pre-
pared9 in good yields using a regiospecific and nucleo-
philic ring opening of chiral epoxides.10 This paper
discloses their application in the synthesis of the title
1′,2′-seconucleophosphonates and reports on the biologi-
cal evaluation of selected acyclophosphonate analogues.
With this in mind, we explored the synthesis of 5′-
phosphonate analogues of 1′,2′-seconucleotides. In pre-
paring the title acyclic analogues, we focused on several
structural features. First, we patterned the spatial
relationship of the C3′- and C4′-positions of our 1′,2′-
seconucleo-5′-phosphonates3 with those of arabino/ribo-
Ch em istr y
The synthetic strategy developed for the preparation
of the title 1′,2′-seconucleophosphonates is depicted in
Scheme 1. This practical, convergent approach uses the
chirons diethyl (3S)-(benzyloxy)-(2R)-hydroxybutane-
phosphonate (1)9 and diethyl (3S),4-bis(benzyloxy)-(2R)-
hydroxybutanephosphonate (2).9 Chloromethylation11
of either 1 or 2 followed by condensation with the
desired, functionalized heterocycle furnished the blocked
phosphonate esters 3-6 and 8. In a typical reaction, 1
was chloromethylated with paraformaldehyde and hy-
drogen chloride gas in dry methylene chloride (CH2Cl2)
at 0 °C. The isolated chloromethyl ether of 1 was then
used immediately with either persilylated cytosine,
thymine, or uracil in the presence of a catalytic amount
of tetraethylammonium iodide. After workup, the pure,
blocked 1′,2′-seconucleophosphates 3-5 were obtained
in good yields.
The synthesis of 6 relied on a different approach and
involved the alkylation of the sodium salt of 6-chloro-
purine12 (generated with sodium hydride) with the
chloromethyl ether of chiron 1 in dry acetonitrile at
room temperature. Thin layer chromatography of the
reaction mixture indicated the presence of two products
presumed to be the N7- and N9-alkylated isomers of
6-chloropurine, with the N9-isomer predominating. The
site of alkylation of the major and faster running isomer
was subsequently confirmed by converting it to the
adenine 1′,2′-seconucleo-5′-phosphonate 11 and examin-
ing the UV spectra of this analogue. Phosphonate 11
exhibited the characteristic spectral features of an N9-
substituted adenine [λmax (pH 1) 257 nm and (pH 11)
259 nm]. Compound 6 was converted to 7 using
methanolic ammonia at 90 °C in a sealed reaction
vessel.
nucleotides, and second, we took into account the
distance between the phosphonate moiety and the
nucleobase ring nitrogen in order to examine what
influence it had on antiviral activity. Regarding the
latter issue, PMEA, (R)-PMPA, (S)-HPMPC, and (S)-
HPMPG, acyclic phosphonate analogues which have the
same distance between the phosphorous and nitrogen
atoms as our title compounds, exhibit potent and
selective antiviral activity.4 Prior to starting our work,
several published accounts described the syntheses of
purine acyclophosphonate nucleotide analogues2,5 using
Arbuzov2 and Wittig5 conditions. Our initial attempt
at preparing 1′,2′-seconucleophosphonates involved the
Arbuzov reaction6 (see Scheme 4) on suitably function-
alized 1′,2′-seconucleosides, e.g., 21.7 We soon realized
that this procedure was cumbersome, time consuming,
and cost inefficient; thus we sought an alternative route.
We envisaged that chiral â- and γ-hydroxybutanephos-
phonate esters would serve as ideal synthons of the
desired chloromethyl ethers which, in turn, could then
be condensed with a variety of nucleobases. This
methodology appeared more attractive, efficient, and
conducive to scale up than our original approach which
Removal of the 3′-O-benzyl protecting group, as well
as the 2′-O-benzyl protecting group in the case of 8, was
carried out smoothly by catalytic transfer hydrogena-
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1130$12.00/0 © 1996 American Chemical Society

1′,2′-Seconucleo-5′-phosphonates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1131
Sch em e 1a
Sch em e 3a
a
Reagents: (a) (CH2O)n, HClg, CH2Cl2, 0 °C; (b) B-TMS,
CH2Cl2, TEAI or B-Na, CH3CN.
Sch em e 2a
a
Reagents: (a) TMS-Br, N2, 0 °C, 1 h; (b) NaOH, EtOH-H2O
(1:1), room temperature; (c) TMS-Br, N2, 0 °C f room temperature,
16 h; (d) TMS-I, N2, 0 °C (1 h) f room temperature (1 h).
Sch em e 4a
a
Reagents: (a) 20% Pd(OH)2/C, EtOH, cyclohexene, reflux (∆).
tion11 using 20% palladium hydroxide on carbon (Pearl-
man’s catalyst) and cyclohexene (Scheme 2). This
debenzylation procedure provided the diethyl phospho-
nate esters 9-12 in excellent yield. Deesterification of
the phosphonate moiety was conducted in several ways
(Scheme 3). Reaction of 9 with 4 equiv of bromotri-
methylsilane (TMS-Br) for 1 h at 0 °C under nitrogen
gave the monoethyl ester 13 in 94% yield, while 10 was
subjected to an aqueous ethanolic solution of sodium
hydroxide at room temperature to furnish a quantitative
yield of the monoethyl ester 14. When the reaction time
of bromotrimethylsilane deesterfication was increased
to ca. 16 h, while keeping the ratio of TMS-Br to diethyl
phosphonate 4:1, complete deesterification of the phos-
phonate moiety was realized. It is worth mentioning
that 5 was deblocked and deesterified in one step to 17
using iodotrimethylsilane.13 The phosphonic acids 15-
18 were easily purified using gravity flow C18 reverse
phase silica gel column chromatography and isolated
in near-quantitative yields.
a
Reagents: (a) Li2NiBr4, THF, room temperature, 15 h; (b)
(CH2O)n, HClg, CH2Cl2, 0 °C; (c) T-TMS, CH2Cl2; (d) (EtO)3P, 155
°C, 28 h or (C6H5O)2POEt, 165 °C, 18.5 h; (e) 10% Pd/C, EtOH,
cyclohexene, reflux (∆), 1.5 h.
deblocking of 3 gave the expected ester 9, the analogous
reaction of the diphenyl ester 22 gave, unexpectedly, the
cyclic phosphonate ester 23. Structure proof of this
compound was derived from the correct elemental
analysis and a comparison of its
1H NMR data with
As mentioned above, our first approach to prepare the
title compounds involved phosphonate formation after
alkylation of the requisite heterocycle (nucleobase). This
approach employed the chloromethyl ether of bromo-
hydrin 20 (Scheme 4). Bromohydrin 20 was easily
prepared by regiospecific opening of epoxide 19 with
dilithium tetrabromonickelate(II).14 Chloromethylation
of 20 followed by condensation with persilylated thy-
mine furnished 21. Introduction of the 5′-phosphonate3
function was accomplished by treatment of the bro-
mothymine 21 with either triethyl or diphenyl ethyl
phosphite to give esters 3 and 22, respectively. While
those of compound 13. While the proton spectrum of
13 in CDCl3 clearly showed three D2O exchangeable
protons, that of 23 had only one corresponding to the
N(3)H proton. Furthermore, in compound 23, the
phosphorus atom is chiral making 23 a mixture of
diastereomers. This was evident from the 1H NMR
spectrum where the 2′-methyl group appeared as two
separate doublets (J ) 6 Hz). On the other hand, the
methyl group in compound 13 appeared, as anticipated,
as one doublet (J ) 6 Hz) since the phosphorus atom is
achiral. The formation of 23 can best be explained by
initial debenzylation followed by intramolecular trans-

1132 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Racha et al.
cooling to room temperature (clear solution), the excess HMDS
was removed under reduced pressure and the residue dried
under high vacuum. A solution of the above chloromethyl
ether in dry CH2Cl2 (20 mL) and tetraethylammonium iodide
(TEAI; ca. 50 mg) were added to the persilylated thymine, and
the mixture was stirred at reflux overnight. The reaction
mixture was then diluted with water (10 mL) and methanol
(30 mL), stirred for 15 min, and evaporated to dryness. The
residue was dissolved in CH2Cl2 (50 mL), washed successively
with water, a 10% Na2S2O3 solution, and brine, and then dried
over anhydrous MgSO4. The viscous material obtained after
solvent removal at reduced pressure was column chromato-
esterification accompanied by the loss of phenol. This
reaction, expectedly, takes place only with phenyl esters
since subjecting the diethyl ester 9 to the same reaction
conditions resulted in the isolation of the starting
material. In addition, when 3 was treated in a similar
manner only debenzylation occurred to furnish the
diethyl phosphonate 9.
Biologica l Eva lu a tion
Compounds 13-18 and 23 were screened for activity
against adenovirus type 2 (AD2), human immunodefi-
ciency virus (HIV), J apanese encephalitis (J BE) virus,
pichinde (PIC) virus, punto toro (PT) virus, Rift Valley
fever (RVF) virus, Sicilian sandfly fever (SFS) virus,
Venezuelan equine encephalomyelitis (VEE) virus, ve-
sicular stomatitis (VSV) virus, and yellow fever (YF)
virus. None of the 1′,2′-seconucleo-5′-phosphonates
exhibited any in vitro activity up to 320 µg/mL. It is
interesting to note that certain thymine, cytosine, and
guanine 2-(phosphonomethyl)-1,3-dioxolane nucleotides,15
which were designed as constrained analogues of PMEA,
and like our title compounds mimic the distance be-
tween the phosphonate moiety and the nucleobase ring
nitrogen, were also shown to be devoid of antiviral
activity. It appears that the pKa of the phosphonate
moiety and thus the spatial location of the oxygen atom
in the acyclic chain, i.e., it must be positioned â to the
phosphorus atom, are critical factors for antiviral
activity.4d
graphed, eluting with ethyl acetate, to give 3 (2.73 g, 95%) as
1
a gum: [R]25 +14.8o (c ) 1.875, CHCl3); H NMR (CDCl3) δ
D
1.16 (d, J ) 6 Hz, 3H, CH3), 1.30 (t, J ) 6 Hz, 6H, 2CH2CH3),
1.70-2.24 (m, 2H, CH2P), 1.84 (s, 3H, CH3), 3.46-4.26 (m, 6H),
4.48 (s, 2H, CH2Ph), 5.24 (ABq, J ) 12 Hz, 2H, OCH2N), 7.18
(br s, 6H, C(6)H, C6H5), 9.92 (s, 1H, NH, D2O exchangeable).
Anal. (C21H31N2O7P) C, H, N, P.
Meth od B: Triethyl phosphite (6.1 mL, 35.2 mmol) was
added to 21 (1.33 g, 3.34 mmol) and the mixture stirred at
155 °C for 28 h. After this period, excess triethyl phosphite
was distilled off and the crude product was column chromato-
graphed. The column was eluted with hexane-ethyl acetate
(1:3) and then ethyl acetate. UV-absorbing fractions contain-
ing 3 were combined and concentrated under diminished
pressure. This procedure furnished pure 3 (1.15 g, 75.6%)
which was identical in all respects with 3 synthesized from
method A.
1-[[(3S)-(Benzyloxy)-1-(diethylphosphonyl)-(2R)-butoxy]-
m eth yl]u r a cil (4). Persilylated uracil [obtained from 2.12 g
(18.96 mmol) of uracil] was coupled as described for 3, with
the chloromethyl ether derived from 1 (3 g, 9.48 mmol), in dry
CH2Cl2 (30 mL) and in the presence of TEAI (70 mg). The
reaction mixture was stirred and heated at reflux for 12 h.
Workup and chromatography (ethyl acetate) afforded pure 4
(3.97 g, 95%): [R]25D -12.4o (c ) 1.00, EtOH); 1H NMR (CDCl3)
δ 1.14 (d, J ) 6 Hz, 3H, CH3), 1.26 (t, J ) 6 Hz, 6H, 2CH2CH3),
1.84-2.54 (m, 2H, CH2P), 3.50-4.36 (m, 6H), 4.54 (s, 2H, CH2-
Ph), 5.22 (ABq, J ) 12 Hz, 2H, OCH2N), 5.62 (d, J ) 8 Hz, 1H,
C(5)H), 7.30 (s, 5H, C6H5), 7.44 (d, J ) 8 Hz, 1H, C(6)H), 9.82
(br s, 1H, NH, D2O exchangeable). Anal. (C20H29N2O7P) C,
H, N, P.
Exp er im en ta l Section
Melting points were determined on a Buchi 535 melting
point apparatus and are uncorrected. Optical rotations were
measured on a Perkin-Elmer Model 141 automatic digital
readout polarimeter. 1H NMR spectra were recorded on either
a Varian EM 390 or a Bruker AM-300 spectrometer using
Me4Si (TMS) as an internal standard. UV absorption spectra
were recorded with a Beckman DU-64 spectrophotometer. All
mositure-sensitive reactions were performed using flame-dried
glassware. Methylene chloride (CH2Cl2) and acetonitrile were
dried over CaH2 and distilled. Evaporations were performed
under diminished pressure using a Buchi rotary evaporator
unless stated otherwise. A Harrison 7924T chromatotron was
used to complete various separations as indicated. Plates (1.0
and 2.0 mm) used were coated with silica gel PF254 containing
CaSO4. Thin-layer chromatography was performed on pre-
coated silica gel plates (60-F254, 0.2 mm) manufactured by
EM Science, Inc., and short-wave ultraviolet light (254 nm)
was used to detect the UV-absorbing compounds. Silica gel
(Merck grade 60, 230-400 mesh, 60 Å) suitable for column
chromatography was purchased from Aldrich. C18 reverse
phase silica gel column chromatography was conducted on
custom bonded Davisil (35-75 µm, 60 Å; Alltech Associates).
All solvent proportions are by volume unless stated otherwise.
Elemental analyses were performed by MHW Laboratories,
Phoenix, AZ.
1-[[(3S)-(Benzyloxy)-1-(diethylphosphonyl)-(2R)-butoxy]-
m eth yl]th ym in e (3). Meth od A: In a three-necked, flame-
dried flask fitted with a gas inlet and a drying tube were added
paraformaldehyde (0.285 g, 9.48 mmol), the alcohol 1 (2.0 g,
6.32 mmol), and dry CH2Cl2 (30 mL). The mixture was cooled
to 0 °C in an ice bath, and dry HCl gas was bubbled into the
solution for 6 h, maintaining the temperature at 0 °C. Calcium
chloride (ca. 5 g) was added cautiously and the mixture stirred
for 15 min. After filtration, the solution was concentrated
under diminished pressure to give the corresponding chloro-
methyl ether, which was directly converted to the title
compound, as follows. Thymine (1.59 g, 12.69 mmol) and
ammonium sulfate (ca. 50 mg) were added to hexamethyl-
disilazane (HMDS; 25 mL). The reaction mixture was stirred
at reflux overnight with the exclusion of moisture. After
1-[[(3S)-(Benzyloxy)-1-(diethylphosphonyl)-(2R)-butoxy]-
m eth yl]cytosin e (5). Persilylated cytosine [obtained from
1.67 g (15 mmol) of cytosine] was coupled as described for 3,
with the chloromethyl ether derived from 1 (3.16 g, 10 mmol),
in dry CH2Cl2 (75 mL) and in the presence of TEAI (75 mg).
The reaction mixture was stirred and heated at reflux for 12
h. Workup and chromatography (ethyl acetate-methanol,
9:1) afforded pure 5 (4.03 g, 92%): [R]25 +9.63o (c ) 0.81,
D
1
EtOH); H NMR (CDCl3) δ 1.14 (d, J ) 6 Hz, 3H, CH3), 1.28
(t, J ) 6 Hz, 6H, 2CH2CH3), 1.82-2.30 (m, 2H, CH2P), 3.52-
4.30 (m, 6H), 4.50 (s, 2H, CH2Ph), 5.04-5.42 (m, 2H, OCH2N),
5.94 (d, J ) 8 Hz, 1H, C(5)H), 7.24 (s, 6H, C(6)H, C6H5), 9.80
(br s, 2H, NH2, D2O exchangeable). Anal. (C20H30N3O6P) C,
H, N, P.
6-Ch lor o-9-[[(3S)-(Ben zyloxy)-1-(d ieth ylp h osp h on yl)-
(2R)-bu toxy]m eth yl]p u r in e (6). To a suspension of sodium
hydride (60%, 0.96 g, 24 mmol; prewashed with petroleum
ether) in dry acetonitrile (170 mL) was added 6-chloropurine
(2.85 g, 18.44 mmol), and the mixture was stirred for 2 h at
room temperature. The chloromethyl ether derived from 1
(5.84 g, 18.46 mmol) was dissolved in acetonitrile (ca. 25 mL)
and added to the above suspension, and the reaction mixture
was stirred for 20 h at room temperature while under dry
nitrogen. At the end of this period, the reaction mixture was
filtered and the filtrate was concentrated in vacuo to a viscous
liquid. This material was column chromatographed, using
acetone-ethyl acetate (2:8) as eluant, to provide 6 (2.48 g, 28%)
as a gum. A slower running material (chromatotron; acetone-
ethyl acetate, 2:8) was isolated and assigned as the N7-isomer
(1H NMR, CDCl3) but was not further characterized. Com-
pound 6 exhibited the following physical data: [R]25 -13.5°
D
1
(c ) 1.15, CH2Cl2); H NMR (CDCl3) δ 1.10 (d, J ) 6 Hz, 3H,
CH3), 1.25 (t, J ) 6 Hz, 6H, 2CH2CH3), 2.00 (ABq, ∆νAB ) 18

1′,2′-Seconucleo-5′-phosphonates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1133
Hz, J AB ) 6 Hz, 2H, CH2P), 3.50-3.65 (m, 1H), 3.80-4.10 (m,
5H), 4.40 (ABq, ∆νAB ) 15 Hz, J AB ) 10 Hz), 6.00 (s, 2H,
OCH2N), 7.10-7.30 (m, 5H, C6H5), 8.50 (s, 1H, C(2)H), 8.80
(s, 1H, C(8)H). Anal. (C21H28O5N4PCl) C, H, N, P, Cl.
9-[[(3S)-(Ben zyloxy)-1-(dieth ylph ospon yl)-(2R)-bu toxy]-
m eth yl]a d en in e (7). Chloropurine 6 (0.360 g, 0.745 mmol)
was dissolved in methanol (3 mL) and placed in a glass liner.
To this solution was added liquid ammonia (7 mL), and the
mixture was heated in a steel reaction vessel at 90 °C for 24
h. After cooling, excess ammonia was vented off and the
remaining residue column chromatographed using ethyl ac-
etate-methanol (95:5) as the eluant to furnish 7 (0.103 g,
30%): [R]25D +4.65° (c ) 0.20, CH2Cl2); 1H NMR (CDCl3) δ 1.05
(d, J ) 6Hz, 3H, CH3), 1.25 (t, J ) 6 Hz, 6H, 2CH2CH3), 2.00
(ABq, ∆νAB ) 18 Hz, J AB ) 6 Hz, 2H, CH2P), 3.33-3.60 (m,
1H), 3.70-4.30 (m, 5H), 4.45 (s, 2H, OCH2Ph), 5.70 (s, 2H,
OCH2N), 6.30-6.60 (br s, 2H, NH2, D2O exchangeable),
7.10-7.30 (m, 5H, C6H5), 7.95 (s, 1H, C(2)H), 8.30 (s, 1H,
C(8)H). Anal. (C21H30O5N5P) H, N; C: calcd, 54.42; found,
54.98.
4.60 (m, 10H, 2H D2O exchangeable), 4.98-5.62 (m, 2H,
OCH2N), 7.34 (br s, 1H, C(6)H), 9.98 (br s, 1H, NH, D2O
exchangeable). Anal. (C14H25N2O8P) C, H, N.
1-[[1-(Eth oxyh yd r oxyp h osp h in yl)-(3S)-h yd r oxy-(2R)-
bu toxy]m eth yl]th ym in e (13). A solution of 9 (0.34, 0.92
mmol) and TMS-Br (0.56 g, 3.68 mmol) in dry CH2Cl2 (7.5 mL)
was stirred at 0 °C for 1 h under nitrogen. The volatiles were
removed in vacuo, and the residual oil was taken up in
CH2Cl2 (5 mL) and water (5 mL) and stirred at room temper-
ature for 10 min. The water layer was separated and
evaporated in vacuo, and the residual solid was purified
on a C18 reverse phase column using water as eluant to give
13 (0.29 g, 94%) as a white solid: 1H NMR (D2O)16 δ 1.14 (d,
J ) 6 Hz, 3H, CH3), 1.28 (t, J ) 6 Hz, 3H, CH2CH3), 1.74-
2.35 (m, 2H, CH2P), 7.52 (s, 1H, C(6)H). Anal. (C12H21N2O7P)
C, H, N, P.
1-[[1-(Eth oxyh yd r oxyp h osp h in yl)-(3S)-h yd r oxy-(2R)-
bu toxy]m eth yl]u r a cil (14). To a solution of phosphonate
10 (0.212 g, 0.606 mmol) in 1:1 water and ethanol (3.2 mL)
was added NaOH (0.109 g). The mixture was stirred at room
temperature for 2 h and then neutralized with Dowex 50-H+
ion exchange resin and filtered. The residue obtained after
concentrating the filtrate was dissolved in CH2Cl2 (5 mL) and
water (5 mL). The aqueous layer was washed with CH2Cl2 (2
× 5 mL) and evaporated in vacuo, to afford 14 in quantitative
yield: 1H NMR (D2O)16 δ 1.13 (d, J ) 6 Hz, 3H, CH3), 1.26 (t,
J ) 6 Hz, 3H, CH2CH3), 1.53-2.20 (m, 2H, CH2P), 5.76 (d, J
) 6 Hz, 1H, C(5)H), 7.63 (d, J ) 6 Hz, 1H, C(6)H). Anal.
(C11H19N2O7P‚0.5H2O) C, H, N, P.
1-[[(3S),4-Bis(ben zyloxy)-1-(d ieth ylp h osp h on yl)-(2R)-
bu toxy]m eth yl]th ym in e (8). Persilylated thymine [obtained
from 0.906 g (7.19 mmol) of thymine] was coupled as described
for 3, with the chloromethyl ether derived from 2 (2.02 g, 4.79
mmol), in dry CH2Cl2 (30 mL) and in the presence of TEAI
(30 mg). The reaction mixture was stirred and heated at reflux
for 12 h. Workup and chromatography (ethyl acetate-
methanol, 9:1) afforded pure 8 (2.32 g, 87%): [R]25 +3.97o (c
D
) 1.69, EtOH); 1H NMR (CDCl3) δ 1.25 (t, J ) 6 Hz, 6H,
2CH2CH3), 1.80 (s, 3H, CH3), 1.92-2.38 (m, 2H, CH2P), 3.46-
4.34 (m, 8H), 4.45 (s, 2H, CH2Ph), 4.58 (s, 2H, CH2Ph), 5.18
(ABq, J ) 8 Hz, 2H, OCH2N), 7.24 (br s, 11H, C(6)H, 2C6H5),
9.80 (br s, 1H, NH, D2O exchangeable). Anal. (C28H37N2O8P)
C, H, N, P.
1-[[(3S)-Hyd r oxy-1-p h osp h on yl-(2R)-b u t oxy]m et h yl]-
th ym in e (15). TMS-Br (0.31 g, 2 mmol) was added dropwise,
via syringe, at 0 °C, to the phosphonate 9 (0.180 g, 0.5 mmol)
in CH2Cl2 (10 mL), and the reaction mixture was allowed to
stir for 16 h at room temperature. The volatiles were removed
under vacuum, the residual oil was dissolved in CH2Cl2 (10
mL) and treated with water (10 mL), and the biphasic solution
was stirred at room temperature for 10 min. The aqueous
layer was separated and lyophylized. The residual solid was
purified on a C18 reverse phase column using water as the
1-[[1-(Dieth ylp h osp h on yl)-(3S)-h yd r oxy-(2R)-bu toxy]-
m eth yl]th ym in e (9). A solution of compound 3 (0.560 g, 1.23
mmol) in ethanol (9 mL) and cyclohexene (4.6 mL) was treated
with 20% palladium hydroxide on carbon [Pd(OH)2/C; 50 mg].
The resulting suspension was stirred at reflux for 12 h. After
cooling to room temperature, the mixture was filtered and the
filtrate was concentrated at reduced pressure. The residue
was column chromatographed (ethyl acetate-methanol, 9:1)
to give pure 9 (0.440 g, 100%): [R]25D +19.5° (c ) 1.37, CHCl3);
1H NMR (CDCl3) δ 1.12 (d, J ) 6 Hz, 3H, CH3), 1.32 (t, J ) 6
Hz, 6H, 2CH2CH3), 1.70-2.32 (m, 2H, CH2P), 1.86 (s, 3H, CH3),
3.54-4.36 (m, 7H, 1H exchanges with D2O), 5.24 (ABq, J ) 8
Hz, 2H, OCH2N), 7.32 (br s, 1H, C(6)H), 10.22 (s, 1H, NH, D2O
exchangeable). Anal. (C14H25N2O7P) C, H, N, P.
eluant to furnish 15 (0.147 g, 97%) as a white solid: [R]25
D
+6.67o (c ) 0.51, H2O); H NMR (D2O)16 δ 1.02 (d, J ) 6 Hz,
1
3H, CH3), 1.60-2.12 (m, 2H, CH2P), 1.68 (s, 3H, CH3), 7.36
(br s, 1H, C(6)H). Anal. (C10H17N2O7P) C, H, N.
1-[[(3S)-Hyd r oxy-1-p h osp h on yl-(2R)-b u t oxy]m et h yl]-
u r a cil (16). Compound 16 was prepared from 10 (0.175 g,
0.5 mmol) by the method described for 15, using TMS-Br (0.306
g, 2 mmol), to provide 0.145 g (99%) of 16: [R]25 -15.6° (c )
D
0.91, H2O); 1H NMR (D2O)16 δ 1.16 (d, J ) 6 Hz, 3H, CH3),
2.08 (dd, J ) 18, 6 Hz, 2H, CH2P), 5.86 (d, J ) 7 Hz, 1H, C(5)-
H), 7.74 (d, J ) 7 Hz, 1H, C(6)H). Anal. (C9H15N2O7P) C, H,
N, P.
1-[[1-(Dieth ylp h osp h on yl)-(3S)-h yd r oxy-(2R)-bu toxy]-
m eth yl]u r a cil (10). Compound 10 was prepared from 4 (1.5
g, 3.409 mmol) by the method described for 9 in 97% yield:
1
[R]25 +8.3° (c ) 0.738, EtOH); H NMR (CDCl3) δ 1.06 (d, J
D
1-[[(3S)-Hyd r oxy-1-p h osp h on yl-(2R)-b u t oxy]m et h yl]-
cytosin e (17). Iodotrimethylsilane (TMS-I; 0.679 g, 3.39
mmol) was added dropwise via a syringe at 0 °C to the
phosphonate 5 (0.481 g, 1.095 mmol) in CH2Cl2 (5 mL). The
reaction mixture was stirred under N2 at 0 °C for 1 h and then
at room temperature for 1 h. The volatiles were removed
under reduced pressure, the residual oil was dissolved in CH2-
Cl2 (10 mL) and treated with water (10 mL) containing a few
drops of hydrochloric acid, and the biphasic solution was
stirred at room temperature for 10 min. The aqueous layer
was separated, washed with CH2Cl2 (5 × 5 mL), and lyophi-
lized. The residual solid was purified on a C18 reverse phase
column using water as the eluant to give 17 (0.29 g, 90%):
) 6 Hz, 3H, CH3), 1.26 (t, J ) 6 Hz, 6H, 2CH2CH3), 1.80-2.40
(m, 2H, CH2P), 3.53-4.39 (m, 7H, 1H exchanges with D2O),
5.03-5.50 (m, 2H, OCH2N), 5.70 (d, J ) 9 Hz, 1H, C(5)H),
7.50 (d, J ) 9 Hz, 1H, C(6)H), 10.30 (s, 1H, NH, D2O
exchangeable). Anal. (C13H23N2O7P) C, H, N, P.
9-[[1-(Dieth ylp h osp h on yl)-(3S)-h yd r oxy-(2R)-bu toxy]-
m eth yl]a d en in e (11). Adenine 11 was prepared from 7
(0.274, 0.59 mmol), by the method described for 9, using 20%
Pd(OH)2/C (0.60 g), cyclohexene (4 mL), and ethanol (7.5 mL)
to afford 11 (0.157 g, 71.4%) as a viscous liquid. An analytical
sample was obtained using a chromatotron (ethyl acetate-
methanol, 8:2): [R]25 -2.6° (c ) 1.025, MeOH); 1H NMR
D
(CDCl3) δ 1.10 (d, J ) 6 Hz, 3H, CH3), 1.90-2.20 (m, 2H,
CH2P), 3.70-4.30 (m, 7H, 1H D2O exchangeable), 5.80 (s, 2H,
OCH2N), 6.10-6.40 (m, 2H, NH2, D2O exchangeable), 8.00 (s,
1H, C(2)H), 8.30 (s, 1H, C(8)H). Anal. (C14H24O5N5P) C, H,
N, P.
[R]25 -7.3° (c ) 0.31, H2O); 1H NMR (D2O)16 δ 1.20 (d, J ) 6
D
Hz, 3H, CH3), 2.03 (dd, J ) 15, 6 Hz, 2H, CH2P), 6.26 (d, J )
7.5 Hz, 1H, C(5)H), 7.99 (d, J ) 7.5 Hz, 1H, C(6)H). Anal.
(C9H16N3O6P) C, H, N, P.
1-[[1-(Diet h ylp h osp h on yl)-(3S),4-d ih yd r oxy-(2R)-b u -
toxy]m eth yl]th ym in e (12). Compound 12 was prepared
from 8 (1.8 g, 3.2 mmol) by the method described for 9, using
20% palladium hydroxide on carbon (0.150 g), cyclohexene (16
1-[[(3S),4-Dih yd r oxy-1-p h osp h on yl-(2R)-bu toxy]m eth -
yl]th ym in e (18). Compound 18 was prepared from 12 (0.256
g, 0.67 mmol) by the method described for 15, using TMS-Br
(0.618 g, 4 mmol), to furnish 0.210 g (96%) of 18: [R]25D -10.7o
1
mL), and ethanol (31 mL), to yield 1.18 g (97%) of 12: [R]25
(c ) 0.71, H2O); H NMR (D2O)16 δ 1.88 (s, 3H, CH3), 1.88-
D
-21.5o (c ) 0.66, EtOH); 1H NMR (CDCl3) δ 1.24 (t, J ) 6 Hz,
2.42 (m, 2H, CH2P), 3.46-4.26 (m, 4H), 7.54 (s, 1H, C(6)H).
Anal. (C10H17N2O8P) C, H, N, P.
6H, 2CH2), 1.72-2.54 (m, 2H, CH2P), 1.86 (s, 3H, CH3), 3.34-

1134 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5
Racha et al.
3-O-Ben zyl-1-br om o-(2S,3R)-bu tan ediol (20). To a stirred
solution of the epoxide 19 (6.59 g, 0.037 mol) in 20 mL of dry
THF was added 50 mL of 0.4 N Li2NiBr4 in THF. The reaction
was monitored by TLC, and after 20 min (60 mL) and 15 h
(40 mL), additional quantities of the reagent were added to
the reaction mixture. The reaction mixture was allowed to
stir for another 6 h at room temperature and then poured into
250 mL of pH 7 phosphate buffer. This mixture was extracted
with methylene chloride (8 × 40 mL), and the organic layer
was dried over anhydrous Na2SO4. Evaporation of the organic
layer gave 8.91 g (93%) of 20: 1H NMR (CDCl3) δ 1.20 (d, J )
6 Hz, 3H, CH3), 2.75 (br s, 1H, OH), 3.30-4.12 (m, 4H), 4.47
(ABq, J ) 12 Hz, 2H, OCH2N), 7.25 (s, 5H, C6H5). Anal
(C11H15BrO2) C, H, Br.
Refer en ces
(1) (a) Engel, R. Phosphonates as Analogues of Natural Phosphates.
Chem. Rev. 1977, 77, 349-367. (b) Engel, R. In The Role of
Phosphonates in Living Systems; Hilderband, R. L., Ed.; CRC
Press, Inc.: Boca Raton, FL, 1983; pp 93-138. (c) Krayevsky,
A. A.; Watanabe, K. A. Modified Nucleosides as Anti-Aids
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(2) Reist, E. J .; Sturm, P. A.; Pong, R. Y.; Tanga, M. J .; Sidwell, R.
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as Antiviral Agents. In Nucleotide Analogues as Antiviral Agents;
Martin, J . E., Ed.; American Chemical Society: Washington, DC,
1989; pp 17-34.
(3) In the text, the naming and numbering of the 1′,2′-seconu-
cleophosphonates follow nucleoside nomenclature; however, in
the Experimental Section, the acyclic side chains of the title
compounds are named and numbered in accordance with Chemi-
cal Abstracts Service nomenclature.
(4) (a) Balzarini, J .; De Clercq, E. 5-Phosphoribosyl 1-Pyrophosphate
Synthetase converts the Acyclic Nucleoside Phosphonates 9-(3-
Hydroxy-2-phosphonylmethoxypropyl)- adenine and 9-(2-Phos-
phonylmethoxyethyl)adenine directly to their Antivirally
Active Diphosphate Derivatives. J . Biol. Chem. 1991, 266,
8686-8689. (b) Tsai, C.-C.; Follis, K. E.; Sabo, A.; Beck, T. W.;
Grant, R. F.; Bischofberger, N.; Benveniste, R. E.; Black, R.
Prevention of SIV Infection in Macques by (R)-9-Phosphonyl-
methoxy- propyl)adenine. Science 1995, 270, 1197-1199. (c)
Brodfuehrer, P. R.; Howell, H. G.; Sapino, C., J r.; Vemishetti,
P. A Practical Synthesis of (S)-HPMPC. Tetrahedron Lett.
1994, 35, 3243-3246. (d) Kim, C. U.; Luh, B. Y.; Misco, P. F.;
Bronson, J . J .; Hitchcock, M. J . M.; Ghazzouli, I. Acyclic Purine
Phosphonate Analogues as Antiviral Agents. Synthesis and
Structure-Activity Relationships. J . Med. Chem. 1990, 33, 1207-
1213.
(5) Prisbe, E. J .; Martin, J . C.; McGee, D. P. C.; Barker, M. F.; Smee,
D. F.; Duke, A. E.; Matthews, T. R.; Verheyden, J . P. H.
Synthesis and Antiherpes Virus Activity of Phosphate and
Phosphonate Derivatives of 9-[(1,3-Dihydroxy-2- propoxy)methyl]-
guanine. J . Med. Chem. 1985, 29, 671-675.
(6) Arbuzov, B. A. Michaelis-Arbuzov-Und Perkow Reaktionen.
(Michaelis-Arbuzov-and Perkow Reactions.) Pure Appl. Chem.
1964, 9, 307-335.
(7) Vemishetti, P.; Abushanab, E.; Panzica, R. P. Synthesis of 1′,2′-
Seconucleoside Analogs of AZT. Third Chemical Congress of
North America (ACS), Toronto, Canada, J une 1988; MEDI 17.
(8) This synthetic approach was recently used in the preparation
of the R-enantiomer of ganciclovir phosphonate; see: Chamber-
lain, S. D.; Biron, K. K.; Dornsife, R. E.; Averett, D. R.;
Beauchamp, L.; Koszalka, G. W. An Enantiospecific Synthesis
of the Human Cytomegalovirus Antiviral Agent [(R)-3-((2-Amino-
1,6-dihydro-6-oxo-9H-purine-9-yl)methoxy)-4-hydroxybutyl]phos-
phonic Acid. J . Med. Chem. 1994, 37, 1371-1377.
(9) Li, Z.; Racha, S.; Dan, L.; El-Subbagh, H. I.; Abushanab, E. A
General and Facile Synthesis of â- and γ-Hydroxy Phosphonates
from Expoxides. J . Org. Chem. 1993, 58, 5779-5783.
(10) (a) Abushanab, E.; Vemishetti, P.; Leiby, R. W.; Singh, H. K.;
Mikkilineni, A. B.; Wu, D. C.-J .; Racha, S.; Panzica, R. P. The
Preparation of Chiral Butanetriols and -tetrols. J . Org. Chem.
1988, 53, 2598-2602. (b) Vargeese, C.; Abushanab, E. An
Efficient Synthesis of 2-Deoxypentofuranoses. J . Org. Chem.
1990, 55, 4400-4403.
(11) (a) Vemishetti, P.; Leiby, R. W.; Abushanab, E.; Panzica, R. P.
A Practical Synthesis of Ethyl 1,2,4-Triazole-3-Carboxylate and
its use in the Formation of Chiral 1′,2′-Seconucleosides of
Ribavirin. J . Heterocycl. Chem. 1988, 25, 651- 654. (b) Vem-
ishetti, P.; Saibaba, R.; Panzica, R. P.; Abushanab, E. Prepara-
tion of 2′-Deoxy-2′-fluoro-1′,2′-seconucleosides as Potential An-
tiviral Agents. J . Med. Chem. 1990, 33, 681-686.
(12) Abushanab, E.; Sarma, M. S. P. 1′,2′-Secodideoxynucleosides as
Potential Anti-HIV Agents. J . Med. Chem. 1989, 32, 76-79.
(13) Olah, G. A.; Narang, S. C. Iodomethylsilane-A Versatile Syn-
thetic Reagent. Tetrahedron 1982, 38, 2226-2277 and references
cited therein.
1-[[(3S )-(Be n zyloxy)-1-b r om o-(2R )-b u t oxy]m e t h yl]-
th ym in e (21). Coupling of persilylated thymine [obtained
from 3.0 g (0.0024 mol) of thymine] was accomplished in a
similar manner as reported for 3 with 7.10 g (81.7% pure, 0.019
mol) of (3S)-(benzyloxy)-1-bromo-(2R)-(chloromethoxy)butane,
all of which was dissolved in 60 mL of dry methylene chloride
and in the presence of a catalytic amount of TBAI. This
procedure afforded 21 in quantitative yield after column
chromatography using ethyl acetate-hexane (1:1) as eluant.
Fractions containing 21 were pooled and concentrated, and
on standing the product crystallized: mp 51-56 °C; [R]25D +7.6
1
(c ) 1.81, EtOH); H NMR (CDCl3) δ 1.15 (d, J ) 6 Hz, 3H,
CH3), 1.88 (s, 3H, CH3), 3.42-4.00 (m, 4H), 4.48 (d, 2H, OCH2-
Ph), 5.52 (s, 2H, OCH2N), 7.09 (s, 1H, C(6)H), 7.39 (s, 5H,
C6H5), 9.78 (br s, 1H, NH, D2O exchangeable). Anal. (C17H21
-
BrN2O4) C, H, Br, N.
Dip h en yl 1-[[(3S)-(Ben zyloxy)-(2R)-b u t oxy]m et h yl]-
th ym in e-1′-p h osp h on a te (22). Diphenyl ethyl phosphite
(3.2 g, 12.21 mmol) and 21 (1.2 g, 3.02 mmol) were combined
and heated at 165 °C for 18.5 h. The excess reagent was
distilled off, and the residue was purified by (silica gel, 60-
230 mesh) column chromatography. Elution with hexene-
ethyl acetate (1:1) gave 0.28 g (23%) of starting material 21
and 0.65 g (39.1%) of the phosphonate 22, as a viscous
1
material: [R]25 +15.8 (c ) 1.51, CHCl3); H NMR (CDCl3) δ
D
1.14 (d, J ) 6Hz, 3H, CH3), 1.63 (s, 3H, CH3), 2.03-2.72 (m,
2H, CH2P), 3.55-3.90 (m, 1H), 3.93-4.35 (m, 1H), 4.48 (d, 2H,
OCH2Ph), 5.13 (ABq, J ) 10.5 Hz, 2H, OCH2N), 6.85-7.38
(m, 16H, C6H5, C(6)H), 9.10 (br s, 1H, NH, D2O exchangeable).
Anal. (C29H30N2O7P) C, H, N, P.
P h en yl 1-[[(3S)-Hyd r oxy-(2R)-bu toxy]m eth yl]th ym in e
1′,3′-Cyclic P h osp h on a te (23). Catalytic transfer hydroge-
nation of the phosphonate 22 (0.87 g, 1.58 mmol) which was
dissolved in 46.6 mL of absolute ethanol was carried out, as
described earlier with the exception that 10% Pd/C (0.78 g)
was used as catalyst in the presence of 23.3 mL of cyclohexene
at reflux for 1.5 h. Purification of product by column chro-
matography (silica gel, 60-230 mesh) using ethyl acetate as
eluant afforded 0.375 g (64.7%) of pure 23, as a gummy
material: 1H NMR (CDCl3) δ 1.23 and 1.37 (2 d, J ) 6 Hz,
3H, CH3), 1.87 (s, 3H, CH3), 2.08-2.70 (m, 2H, CH2P), 3.96-
4.88 (m, 2H), 5.06-5.28 (2 ABq, 2H, OCH2N), 6.90-7.47 (m,
6H, C6H5, (C(6)H), 9.13 (br d, 1H, NH, D2O exchangeable).
Anal. (C16H19N2O6P) C, H, N, P.
In Vitr o An tivir a l Assa ys:17-20 (a ) In h ibition of Cyto-
p a th ic Effect (CP E). Virus was absorbed for 1 h in 96-well
monolayer cultures of Vero cells (PT, J BE, SFS, YF), H.Ep.2
cultures (AD2), or LLC-MK2 cells (PT), after which tissue
culture medium containing various drug concentrations was
added. At the day of maximum CPE in virus control wells,
medium was removed and monolayers were stained with
crystal violet for microscopic CPE determination.
(b) In h ibition of Vir u s P la qu e F or m a tion . RVF plaque
reduction was determined by adding a semisolid agarose
overlay containing various drug concentrations to Vero mono-
layers after adsorption with 40-100 pfu of RVF virus. After
96 h, the overlay was removed and plaques were visualized
by crystal violet staining of the monolayers.
(14) Dawe, R. D.; Molinski, T. F.; Turner, J . V. Dilithium Tetrabro-
monickelate (II) as
Reaction with Epoxides. Tetrahedron Lett. 1984, 25, 2061-
2064.
a Source of Soft Nucleophilic Bromide:
(15) Bednarski, K.; Dixit, D. M.; Mansour, T. S.; Colman, S. G.;
Walcott, S. M.; Ashman, C. Synthesis of Racemic 2-Phospho-
nomethyl-1,3,-dixoloane Nucleoside Analogues as Potential
Antiviral Agents. Bioorg. Med. Chem. Lett. 1995, 5, 1741-
1744.
(16) The 1H NMR spectra of 13-16 and 18 were run in D2O. In
addition to other proton resonances, the signal(s) for the OCH2N
protons were not completely visible due to overlap with the HDO
peak.
Ack n ow led gm en t. We thank Dr. V. P. Pragna-
charyulu and Mrs. Rena Fullerton for technical as-
sistance.

1′,2′-Seconucleo-5′-phosphonates
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 5 1135
(17) Kirsi, J . J .; North, J . A.; McKernan, P. A.; Murray, B. K.;
Canonico, P. G.; Huggins, J . W.; Srivastava, P. C.; Robins, R. K.
Broad-Spectrum Antiviral Activity of 2-â-D-Ribofuranosyl-4-
(19) Huggins, J . W.; Robins, R. K.; Canonico, P. G. Synergistic
Antiviral Effects of Ribavirin and the C-Nucleoside Analogs
Tiazofurin and Seleazofurin Against Togaviruses, Bunyaviruses,
and Arenaviruses. Antimicrob. Agents Chemother. 1984, 26,
476-480.
Carboxamide,
a New Antiviral Agent. Antimicrob. Agents
Chemother. 1983, 24, 353-361.
(18) Kirsi, J . J .; McKernan, P. A.; Burns, N. J ., III; North, J . A.;
Murray, B. K.; Robins, R. K. Broad-Spectrum Synergistic
Antiviral Activity of Selenazofurin and Ribavirin. Antimicrob.
Agents Chemother. 1984, 26, 466-475.
(20) Testing was conducted under the auspices of USAMRIID.
J M9506783