Synthesis and antiviral activity of pyranosylphosphonic acid nucleotide analogues

Article abstract of DOI:10.1021/jm950788+

Pyranosyl nucleotide analogues have been designed so that the intramolecular base to phosphorus distance closely approximates that of natural nucleotides. This was achieved by attaching the phosphorus directly at the anomeric position and the base at the

Full text of DOI:10.1021/jm950788+

J . Med. Chem. 1996, 39, 1321-1330
1321
Syn th esis a n d An tivir a l Activity of P yr a n osylp h osp h on ic Acid Nu cleotid e
An a logu es
Petr Alexander,† V. V. Krishnamurthy, and Ernest J . Prisbe*
Gilead Sciences, Inc., 353 Lakeside Drive, Foster City, California 94404
Received October 26, 1995X
Pyranosyl nucleotide analogues have been designed so that the intramolecular base to
phosphorus distance closely approximates that of natural nucleotides. This was achieved by
attaching the phosphorus directly at the anomeric position and the base at the 4-position of
the carbohydrate. A series of compounds incorporating natural bases and having this novel
structure were made via a short synthesis starting from commercially available glycals.
Addition of triisopropyl phosphite to the glycals furnished R- and â-2-enopyranosylphosphonates
which were then substituted with the heterocycle using Mitsunobu chemistry. Deprotection
afforded the 2′,3′-unsaturated isonucleotide analogue. In some cases the deprotection sequence
induced double-bond migration leading to the 1′,2′-unsaturated derivative. NMR spectroscopic
structural analysis established an axial preference for the base and an equatorial preference
for the â-phosphorus which results in intramolecular base to phosphorus distances within 1 Å
of that of natural nucleotides. All of the deprotected compounds were screened for inhibition
of HCMV, HSV-2, and HIV replication. Several compounds inhibited HCMV and HSV-2, the
most potent of which was the unsaturated cytosine analogue 18 (HCMV IC50 ) 10 µM, HSV-2
IC50 ) 85 µM). None of the compounds were cytotoxic at the highest dose (1 mM) tested. None
of the compounds were inhibitory to HIV.
Biologically active pyranosyl nucleosides have been
known for over 40 years.1 Early examples such as
gougerotin, blasticidin S, amicetin, and their relatives
are elaborated by Streptomyces, inhibit protein synthe-
sis, and have antibacterial activity. However, examples
of pyranosyl nucleosides with significant antiviral activ-
ity are much rarer. This lack of activity might be
considered surprising in light of the structural diversity
of nucleoside antiviral agents. For instance, the most
frequently utilized target for the intervention of herpes
virus and human immunodeficiency virus (HIV) replica-
tion is their DNA polymerases. Some inhibitors of these
enzymes are the triphosphate derivatives that are
generated intracellularly from a wide structural range
of nucleoside analogues.2 This range includes analogues
in which the furanose has been replaced by an acyclic
moiety, cyclopentane, cyclobutane, oxetane, carbon-
branched furanose, dioxolane, oxathiolane, and tetrahy-
drothiophene. The fact that pyranosyl nucleosides are
not on this list demonstrates an obvious incompatibility
of the pyranose. On the other hand, pyranosyl nucleo-
sides have recently been reported that have the hetero-
cyclic base transposed from the 1′- to the 2′-position on
the pyranose which results in significant antiherpes
activity.3 Thus, the presence of a pyranose may be a
detriment to activity only when the base and sugar are
oriented in the traditional sense, i.e., a â-D-purinyl- or
-pyrimidinylpyranoside.
F igu r e 1. (4-Cytosinyl-2,3,4-trideoxy-â-D-threo-hex-2-enopy-
ranosyl)phosphonic acid (black) superimposed on dCMP (gray).
phosphate is eventually appended be similar to that of
natural furanose nucleosides. This distance is expected
to be an important determinant in successful phospho-
rylation as well as in subsequent binding to the poly-
merase.4 A simple modeling comparison of energy-
minimized structures5 showed a good fit (rms ) 0.35 Å
for phosphate and heterocyclic moieties) with the natu-
ral nucleotides if a pyranose was substituted with a
phosphonic acid at the 1-position and with the hetero-
cyclic base at the 4-position. Figure 1 is the superim-
position of dCMP (gray) with one such example, 4-cy-
tosinyl-2-enopyranosylphosphonic acid (black). Thus,
these isonucleotides became the target of our synthetic
endeavors. To our knowledge, nucleotide analogues in
which the phosphorus occupies an anomeric position are
unprecedented.
Our antiviral design strategy was based on this
premise. We chose to use the pyranose ring as a scaffold
to position the key recognition elements advantageously,
with slight regard to their natural positions. The
primary criterion was that the distance between the
heterocyclic base and the position at which the tri-
Additional incentive for the synthesis of such com-
pounds was provided by a number of advantages we
predicted to be inherent in these structures. For
instance, glycosidic bond cleavage is a frequently en-
† Current address: Institute of Organic Chemistry and Biochemis-
try, Academy of Sciences of the Czech Republic, Prague, Czech
Republic.
X Abstract published in Advance ACS Abstracts, February 1, 1996.
0022-2623/96/1839-1321$12.00/0 © 1996 American Chemical Society

1322 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Alexander et al.
Sch em e 1a
Sch em e 2a
a
a
Reagents: (a) P(OiPr)3, BF3‚Et2O, toluene; (b) NH3/MeOH; (c)
Reagents: (a) B, Ph3P, DEAD, THF; (b) NH3/MeOH or NEt3/
MeOH; (c) 80% AcOH; (d) TMSBr, lutidine, CH3CN.
TrCl, pyridine.
Ferrier rearrangement.9 Trialkyl phosphites have also
been added to both acyclic and cyclic enol ethers, but
this reaction is performed with HCl and proceeds via
an Arbuzov type mechanism.10 We have found that
trialkyl phosphites also are effective participants in the
Ferrier rearrangements, affording unsaturated pyran-
osylphosphonates in high yields under mild conditions.
Thus, commercially available tri-O-acetyl-D-glucal (1) on
treatment with triisopropyl phosphite in the presence
of catalytic boron trifluoride diethyl etherate furnished
the 2,3-unsaturated pyranose 2 in 93% yield. The
product, isolated as an approximately equal mixture of
anomers, was deacetylated using either potassium
carbonate in methanol or ammonia-saturated methanol.
Compounds 2 and 3 â have been previously described
but as the dimethyl rather than the diisopropyl esters.9
After selective tritylation of the primary hydroxyl group,
chromatographic separation on silica gel afforded an
overall yield from 1 of 46% for the â-anomer 4 and 39%
for the R-anomer 5. The assignment of anomeric
configuration was based on NMR spectroscopic methods
and is discussed below.
Having the pyranosylphosphonates in hand, the
heterocyclic bases were next appended at the 4-position.
Scheme 2 illustrates the synthetic pathway starting
from the â-anomer. Unsuccessful attempts to displace
a methanesulfonate at the 4-position with the hetero-
cycles prompted trials using Mitsunobu chemistry.11
Protected derivatives of cytosine, thymine, adenine, and
guanine were reacted with the 4-hydroxy sugar in the
presence of triphenylphosphine and DEAD. In all cases
the desired compound, having the inverted configuration
at the sugar 4-position, was the major reaction compo-
nent.
countered, degradative pathway of nucleoside antivirals,
particularly the 2′,3′-dideoxynucleosides.6 The place-
ment of the heterocyclic base at the 4-position of the
pyranose rather than at the anomeric position would
alleviate this deficiency. Nucleoside analogues require
stepwise phosphorylation to their 5′-triphosphate de-
rivative before they can interact with viral polymerases.
The a priori inclusion of the phosphonate would allow
these new analogues to bypass the first, often rate-
limiting, phosphorylation step. The presence of the
pyran ring oxygen â to the phosphorus would be
expected to increase the pKa of the phosphonic acid
making it isoelectronic with a phosphate.7 Lastly, a
number of acyclic, phosphonate, nucleotide analogues
exemplified by 9-[2-(phosphonylmethoxy)ethyl]adenine
(PMEA) and 1-[3-hydroxy-2-(phosphonomethoxy)propyl]-
cytosine (HPMPC), have significant antiviral activity.8
In the proposed pyranosyl nucleotides, the linkage
between the base and phosphorus along the sequence
4′,5′,O5′,1′ is the same as is present in HPMPC and
PMEA.
In this paper we describe an efficient synthesis of
these new analogues from commercially available gly-
cals. Some of the compounds were inhibitors of HSV-2
and CMV. None showed cytotoxicity at 1 mM concen-
trations, the highest dose tested.
Ch em istr y
Construction of the requisite nucleotide analogues
followed a common strategy. Readily available glycals
were converted to 2,3-unsaturated glycopyranosylphos-
phonates which were then substituted at the 4-position
with the purine or pyrimidine base. These intermedi-
ates were either directly deprotected or further modified
in order to provide the 1,2-unsaturated isomer or fully
saturated analogues.
The synthesis of the first key intermediate, the
differentially protected (2,3-dideoxyhex-2-enopyranosyl)-
phosphonate, is illustrated in Scheme 1. Paulsen has
reported the addition of dialkyl phosphites to peracety-
lated glycals in the presence of boron trifluoride to
efficiently furnish 2-enopyranosylphosphonates via a
The deprotection of the compounds proceeded through
a common sequence of reactions. The purine or pyrim-
idine moieties were first deacylated with ammonia-
saturated methanol or a solution of triethylamine in
methanol. The latter method sometimes afforded fewer
side products. The trityl group was removed next by
heating in aqueous acetic acid, and lastly, the phospho-
nate esters were cleaved with bromotrimethylsilane in

Pyranosylphosphonic Acid Nucleotide Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1323
Sch em e 3a
Sch em e 4a
a
Reagents: (a) P(OiPr)3, BF3‚Et2O, toluene; (b) NH3/MeOH; (c)
TrCl, pyridine; (d) N3BzC, Ph3P, DEAD, THF; (e) 80% AcOH; (f)
TMSBr, lutidine, CH3CN.
in this case, are presumably initiated by the abstraction
of the anomeric proton.
Thus, a series of 1′-ene derivatives were produced
along with the targeted 2′-ene compounds. The cytosine
analogue was the most troublesome, affording exclu-
sively the 1′-olefin during any attempt to remove the
N4-benzoyl protecting group. Similarly, attempts to
remove the N3-benzoyl group on thymine analogue 23
with methanolic ammonia caused complete 2′- to 1′-
double-bond migration, although this could be mitigated
by substituting triethylamine for the ammonia. Pre-
dictably, all efforts to convert the 6-chloropurine sub-
stituent on 24 to adenine, using ammonia, led to double-
bond migration. However, the adenine analogue 40
could eventually be derived using N6-phenoxyacetyl
protection (25) which successfully succumbed to removal
with methanolic triethylamine. In the case of the
guanine analogue, an approximately equal amount of
the rearranged olefin 30 was produced along with the
deacylated 2′-unsaturated 41. Fortunately, the remain-
ing hydroxyl and phosphonic acid deprotections required
in this series proceeded uneventfully as before.
a
Reagents: (a) B, Ph3P, DEAD, THF; (b) NH3/MeOH or NEt3/
MeOH; (c) 80% AcOH; (d) TMSBr, lutidine, CH3CN.
the presence of lutidine. The final enopyranosylphos-
phonic acids were purified by a combination of ion
exchange and reverse phase chromatography.
The key to the success of this reaction sequence was
the proper selection of the heterocyclic protecting group.
Without protection, the Mitsunobu coupling proceeded
very poorly or not at all. Acyl protection of the hetero-
cycle facilitated the reaction, but the subsequent basic
cleavage conditions in some cases led to complex product
mixtures, the purine derivatives being particularly
vulnerable. Therefore, removal of the benzoyl group at
N3 of the thymine derivative was unsuccessful with
ammonia/methanol but satisfactory with 10% triethyl-
amine in methanol. Adenine and guanine required the
use of the labile phenoxyacetyl and diphenylcarbamoyl
protecting groups, respectively.
In order to investigate the effect of the anomeric
configuration on antiviral activity, the same sequence
of reactions was carried out starting from the R-pyran-
osylphosphonate 5 (Scheme 3). However, working with
the R-anomers was problematic because it was discov-
ered that this series was much more susceptible to base-
induced, double-bond migration than was the â-series.
The basic conditions required for the removal of acyl
protecting groups was often enough to completely drive
the 2′-position double bond to the 1′-position.12 Such
rearrangements of allylphosphonates to vinylphospho-
nates under basic conditions are well precedented13 and,
Other analogues were made starting from commer-
cially available tri-O-acetyl-L-glucal (48) and di-O-
acetyl-L-rhamnal (57). From L-glucal, only the cytosine
analogue was made as shown in Scheme 4. The
synthesis paralleled that used in the D-series with the
exception that the mixture of phosphonyl sugar anomers
were not separated but, instead, carried through the
ammonia and acetic acid deprotection steps. Product
separation at this point provided the â-diisopropylphos-
phonate 52 and the glycal diisopropylphosphonate 55.
Apparently, all of the R-anomer had again undergone
rearrangement to the glycal under the basic conditions.

1324 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Alexander et al.
Sch em e 5a
bromotrimethylsilane afforded the saturated product 73
in 88% overall yield.
Str u ctu r a l Elu cid a tion s
With the compounds in hand, NMR was first relied
upon to confirm structures and deduce the configura-
tions of the substituents on the pyranose ring. Using
this information, a computer model was constructed
from which intramolecular distances were measured
and compared with those of natural nucleosides. In this
way, the three-dimensional similarity of the target
compounds and natural nucleotides could be assessed.
Structural assignments of the products were based
on 1D and 2D NMR spectra. In general, the molecular
1
skeleton was first established using H-1H spin net-
work as determined by COSY spectra and carbon
1
connectivity based on one-bond and two/three-bond H-
13C correlation from HMQC14 and HMBC15 spectra,
respectively. Analysis of the COSY spectra established,
in addition to the proton-coupling network in the
pyranose ring, the position of the double bond (2′-3′ or
1′-2′) in the ring. The 13C chemical shifts of the
protonated carbons were first assigned from the 1H-
13C HMQC spectra; then the HMBC spectra were
analyzed to establish the molecular skeleton. The
following diagnostic three-bond correlations were ob-
served. (i) H1′-C5′ and H5′-C1′ correlations estab-
lished the ether linkage between the 1′- and 5′-carbons.
(ii) The correlation between the base proton (H8 in the
case of guanine and adenine and H6 in the case of
cytosine and thymine) and C4′ established the location
of the base at the 4′-position. (iii) In the case of purine
derivatives, the C4 carbon of the base shows a correla-
tion to H8 of the base and H4′ of the sugar, establishing
the sugar-base connectivity through N9 and not N7.
Next, ring conformation and relative stereochemistry
of the base and the anomeric center were determined
by examination of either 1D NOE difference spectra or
2D 1H-1H NOESY spectra. In all cases, the base proton
(H6 in the case of cytosine and thymine and H8 in the
case of guanine and adenine) shows a NOE to H6′ (in
addition to H4′) and not to H5′. However, H4′ shows a
NOE to both H5′ and H6′. This indicates that while
the base is syn with respect to C6′ and anti with respect
to H5′, H4′ is syn with respect to both C6′ and H5′.
Therefore, C6′ and H4′ are in an equatorial position,
and the base and H5′ are in axial positions.
With the conformation of the base and H5′ estab-
lished, the anomeric configuration of the 2,3-unsatur-
ated pyranose derivatives was determined based on
NOE interactions with H1′. In one set of isomers,
strong NOE was observed between H1′ and H5′. Since
H5′ had been earlier assigned an axial position, this
strong interaction of H5′ with H1′ establishes the
anomeric configuration to be â (with the H1′ in the axial
and the phosphorus in the equatorial positions). In the
other set of isomers, a weak, long range NOE is observed
between the base proton (H6 in the case of cytosine and
thymine and H8 in the case of guanine and adenine)
and H1′ and no NOE is observed between H5′ and H1′.
Since the base had been earlier assigned to the axial
position (and C1′-C2′-C3′-C4′ is coplanar), its interac-
tion with H1′ establishes the anomeric configuration to
be R with H1′ in the equatorial and the phosphorus in
the axial positions.
a
Reagents: (a) P(OiPr)3, BF3‚Et2O, toluene; (b) NH3/MeOH; (c)
B, Ph3P, DEAD, THF; (d) TMSBr, lutidine, CH3CN.
Sch em e 6a
a
Reagents: (a) Pd/C, H2, MeOH; (b) TMSBr, lutidine, CH3CN.
Removal of the phosphonate esters with bromotrimeth-
ylsilane afforded the (cytosinyl-L-pyranosyl)phosphonic
acids 53 and 56.
Scheme 5 illustrates the reaction sequence used to
synthesize analogues from L-rhamnal. In general, yields
were higher than those obtained with the 6-hydroxyl
sugars. As had been done with L-glucal, phosphonate
anomers were carried as a mixture into the ammonia
treatment step. Different enopyranosyl isomers were
obtained depending on the heterocycle present. The
conversion of 6-chloropurine to adenine led entirely to
rearrangement and the glycal phosphonate 68. The
debenzoylation of the cytosine analogue 59 gave the
desired â-enopyranosylphosphonate 62 plus the glycal
phosphonate 66, while benzoyl removal from thymine
analogue 60 also led to the glycal phosphonate 67 as
well as the â-enopyranosylphosphonate 63. This varied
collection of intermediate phosphonates were all dees-
terified with bromotrimethylsilane to furnish the phos-
phonic acids shown.
Also for biological comparison purposes, the fully
saturated (cytosinylpyranosyl)phosphonic acid 73 was
made (Scheme 6). Hydrogenation of 14 over 10%
palladium on carbon followed by deesterification with

Pyranosylphosphonic Acid Nucleotide Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1325
Ta ble 1. Inhibitory Effects of Compounds on HCMV
Replication, HSV-Induced Cytopathogenicity, and Host Cellular
Growth
F igu r e 2. NMR-indicated conformations of the pyranosyl
isonucleotides: (a) â-2′-ene, (b) R-2′-ene, (c) 1′-ene, and (d)
â-saturated.
In the case of 1,2-unsaturated pyranose derivatives,
one of the 3′-protons shows a NOE to the base proton
(H8 purines, H6 pyrimidines) and H4′, while the other
shows an interaction with H4′ and H5′. Hence, they
are assigned to the equatorial and axial H3′ protons,
respectively. These NOE interactions of the H3′ protons
are again consistent with the heterocyclic base being
in the axial position.
a
Concentration of compound required to achieve a 50% reduc-
As determined by the NMR studies, the substituents
on the pyranoses consistently adopted the same con-
figurational pattern. The purine or pyrimidine base was
always axial, the hydroxymethyl or methyl substituent
at 5′ was equatorial, and the phosphorus was equatorial
when â and axial when R. The generalized structures
are depicted in Figure 2. Thus, the spatial positioning
of substituents was the same for all â-anomers and
likewise for all the R-anomers.
b
tion in the replication of HCMV (Towne) in NHDF cells. Con-
centration of compound required to achieve 50% protection of
MRC-5 cells against cytopathic effect of HSV-2 (E 194). c Concen-
tration of compound required to reduce NHDF cell growth by 50%.
d
Selectivity index ) CC50 (NHDF)/IC50 (HCMV).
Biologica l Resu lts
Having met the primary synthetic objective of posi-
tioning the base and phosphorus to closely approximate
the spatial relationship present in natural nucleotides,
the compounds were then tested for biological activity.
The in vitro antiherpes activities of the nucleotide
analogues are shown in Table 1. Compounds were
tested for their inhibition of the replication of HCMV
(Towne strain) in NHDF cells and their inhibition of the
cytopathogenicity of HSV-2 (E 194 strain) in MRC-5
cells. Estimates of toxicity were obtained by determin-
ing the 50% cytotoxic concentration for mock-infected
NHDF cells. HPMPC was included as a control.
Some of the compounds in this series displayed
moderate inhibition of HCMV and HSV-2. The most
active example, (4-cytosinyl-2,3,4-trideoxy-â-D-threo-
hex-2-enopyranosyl)phosphonic acid (18), was ca. 20-
fold less active than the control compound HPMPC.
However, no toxicity was observed for 18 up to the
highest concentration tested of 1 mM.
In order to estimate intramolecular distances, we
relied on a minimization program5 to construct a low-
energy model. Compound 18, having the greatest
antiviral activity of the series, was chosen as the
example. The completed minimization qualitatively
matched the NMR results in configuring the base,
phosphorus, and hydroxymethyl substituents axially,
equatorially, and equatorially, respectively, on the pyra-
nose (as shown in both Figures 1 and 2a). This
concurrence provided confidence that the minimized
structure was a reasonable approximation of the actual
solution conformation. Using the computer-generated
model, intramolecular atomic distances were measured
and found to compare favorably with the analogous
distances in the natural nucleotides. The calculated
distance from the N1 of the base to phosphorus is 5.0
Å. This is similar to the N9-P distances of 5.45 and
4.36 Å reported for two different crystal forms of
AMP.16,17 Also, the measured distance between N1 and
C1′, which is the carbon that would correspond to the
position of C5′ of natural nucleotides, is 3.8 Å. The
distance between N1 and C5′ of CMP in the solid state
is reported to be 4.2 Å.18
The results elucidated some general trends in struc-
ture-activity relationships. Analogues containing cy-
tosine were the most active. D-Isomers were more active
than L-isomers. Compounds having 2,′3′-unsaturation
were more active than the analogous 1′,2′-unsaturated
compounds which, in the single example shown, were
more active than the fully saturated analogue. None

1326 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Alexander et al.
4.7-4.9 (m, 2H), 5.88 (m, 1H), 6.02 (m, 1H), 7.2-7.5 (m, 15H).
Anal. (C31H37O6P‚0.5 CH2Cl2) C, H.
of the compounds were toxic to NHDF cells at the
highest concentrations tested.
The compounds were also screened for inhibition of
HIV (III b) in MT2 cells. None had activity.
5: 1H NMR (CDCl3) δ 1.2-1.3 (m, 12H), 3.3-3.5 (m, 2H),
3.53 (m, 1H), 4.22 (m, 1H), 4.53 (d, J ) 17.6 Hz, 1H), 4.76 (m,
2H), 5.9-6.0 (m, 2H), 7.2-7.4 (m, 15H). Anal. (C31H37O6P‚0.5
CH2Cl2) C, H.
Diisop r op yl [4-(N4-Ben zoylcytosin -1-yl)-2,3,4-tr id eoxy-
6-O-(tr iph en ylm eth yl)-â-D-th r eo-h ex-2-en opyr an osyl]ph os-
p h on a te (6). Diethyl azodicarboxylate (0.522 g, 3.0 mmol)
was added dropwise over 5 min to a 0 °C stirred suspension
of 4 (1.8 g, 2.0 mmol), N4-benzoylcytosine19 (0.645 g, 3.0 mmol),
and triphenylphosphine (0.786 g, 3 mmol) in anhydrous THF
(20 mL) under nitrogen. The mixture was stirred at 0 °C for
4 h and then at room temperature for 20 h before being diluted
with EtOH (5 mL) and concentrated in vacuo. The residue
was partitioned between ethyl acetate (50 mL) and water (50
mL), and the aqueous layer was extracted further with ethyl
acetate (50 mL). The combined organic phases were dried
(Na2SO4), filtered, and concentrated in vacuo affording a
viscous orange-brown oil. Chromatography of the oil on silica
gel (CH2Cl2-MeOH, 98:2) afforded phosphonate 6 (0.557 g,
44%) as a viscous, pale yellow oil: 1H NMR (CDCl3) δ 1.2-1.4
(m, 12H), 3.1-3.3 (m, 2H), 3.92 (m, 1H), 4.62 (m, 1H), 4.8-
4.9 (m, 2H), 5.53 (m, 1H), 5.9-6.1 (m, 2H), 6.42 (d, J ) 7.2,
1H), 7.10-7.60 (m, 20H), 7.92 (d, J ) 7.2, 1H).
Con clu sion
This study has shown that a variety of pyranosyl-
phosphonic acid nucleotide analogues can be readily
constructed from simple glycals. Placement of the
phosphorus at the 1-position and the base at the
4-position approximates the spatial positioning of these
groups in natural nucleotides and in some cases leads
to selective inhibition of herpes virus replication in cell
culture.
Exp er im en ta l Section
Gen er a l Meth od s. Nuclear magnetic resonance spectra
were recorded on a Varian Unity Plus 500 (1H NMR, 500 MHz)
spectrometer, and chemical shifts are reported in parts per
million downfield from external tetramethylsilane. High-
resolution mass spectra (HRMS) were provided by Galbraith
Laboratories, Knoxville, TN, using FAB+. UV spectra of the
samples dissolved in pH 7.5, 5 mM Tris/HCl buffer were
Diisop r op yl
[4-Cyt osin -1-yl-2,3,4-t r id eoxy-6-O-(t r i-
p h en ylm et h yl)-â-D-th r eo-h ex-2-en op yr a n osyl]p h osp h o-
n a te (10). A solution of 6 (0.557 g, 0.76 mmol) in MeOH
saturated at 0 °C with NH3 (20 mL) was kept at room
temperature for 16 h. After concentrating the mixture in
vacuo, the residue was chromatographed on silica gel (CH2-
Cl2-MeOH, 95:5) affording phosphonate 10 (0.38 g, 80%) as a
white solid: 1H NMR (CDCl3) δ 1.3-1.4 (m, 12H), 3.3-3.5 (m,
2H), 4.04 (m, 1H), 4.72 (d, J ) 17.5 Hz, 1H), 5.12 (m, 2H),
5.65 (m, 1H), 5.82 (d, J ) 7.1 Hz, 1H), 6.1-6.3 (m, 2H), 7.1-
7.6 (m, 15H), 7.83 (d, J ) 7.1 Hz, 1H).
recorded on
a Hewlett Packard 8452 spectrophotometer.
Elemental analyses were provided by Galbraith Laboratories.
Column chromatography utilized 70-230 mesh silica gel 60
from E. Merck, while preparative reverse phase HPLC was
performed on a Rainin system using a 2.14 × 25 cm, C-18
column. Melting points were determined on a Thomas Hoover
capillary melting point apparatus and are uncorrected.
Diisop r op yl [4,6-Di-O-a cetyl-2,3-d id eoxy-r(a n d â)-D-
er yth r o-h ex-2-en op yr a n osyl]p h osp h on a te (2). Boron tri-
fluoride etherate (2.4 mL, 20 mmol) was added to a solution
of 3,4,6-tri-O-acetyl-D-glucal (1) (27.2 g, 100 mmol) and triiso-
propyl phosphite (41.6 g, 200 mmol) in 500 mL of dry toluene
under nitrogen. After heating at 100 °C for 4 h, the mixture
was concentrated in vacuo to an oil which was diluted with
toluene (125 mL) and reconcentrated. The concentration from
toluene was repeated thrice more before the residue was taken
up in toluene (800 mL) and extracted with saturated, aqueous
NaHCO3 and water. The organic phase was dried (MgSO4),
filtered, and evaporated affording 2 as a pale yellow oil (35.2
g, 93%): 1H NMR (CDC13) δ 1.1-1.4 (m, 12H), 2.02 (m, 6H),
4.15 (m, 2H), 4.37 (m, 1H), 4.48 (m, 1H), 4.6-4.8 (m, 2H), 5.15
(m, 1H), 5.9-6.0 (m, 2H). Anal. (C16H28O8P) C, H.
Diisop r op yl [2,3-Did eoxy-â(a n d r)-D-er yth r o-h ex-2-
en op yr a n osyl]p h osp h on a te (3). A solution of 2 (18.95 g,
64.4 mmol) in MeOH saturated with NH3 at 0 °C (300 mL)
was stirred at room temperature for 6 h. The solvent was
removed in vacuo and the residue diluted with toluene and
reconcentrated affording 3 as a crude, syrupy, 1:1 mixture of
â- and R-anomers (14.7 g): 1H NMR (CDC13) δ 1.2-1.4 (m,
12H), 3.3 (m, 1H), 3.84 (m, 1H), 4.11 (m, 1H), 4.23 (m, 1H),
4.5 (m, 1H), 4.6-4.8 (m, 2H), 5.8-6.0 (m, 2H). Anal.
(C12H23O6P‚0.25H2O) C, H.
Diisop r op yl (4-Cyt osin -1-yl-2,3,4-t r id eoxy-â-D-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (14). A solution of 10
(0.38 g, 0.6 mmol) in 80% aqueous acetic acid (20 mL) was
stirred at room temperature for 16 h and then concentrated
in vacuo. The residual acetic acid was azeotropically removed
with 5% ethanol in toluene (2 × 50 mL) to give 0.4 g of an oil
which was chromatographed on silica gel (CH2Cl2) to provide
14 (0.195 g, 83%) as a white solid: 1H NMR (CDCl3) δ 1.2-
1.4 (m, 12H), 3.3-3.6 (m, 2H), 3.88 (m, 1H), 4.58 (d, J ) 18
Hz, 1H), 4.7-4.9 (m, 2H), 5.40 (m, 1H), 5.79 (d, J ) 7.2 Hz,
1H), 6.04 (m, 1H), 6.34 (m, 1H), 7.98 (d, J ) 7.2 Hz, 1H).
(4-Cyt osin -1-yl-2,3,4-t r id eoxy-â-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (18). Bromotrimethylsilane (0.66
mL, 5.0 mmol) was added dropwise to a solution of diisopropyl
ester 14 (0.195 g, 0.5 mmol) and 2,4-lutidine (0.6 mL, 5 mmol)
in dry acetonitrile (10 mL) at room temperature under
nitrogen. After stirring for 14 h, the solvents were removed
in vacuo and the residual, yellow oil was first taken in dry
acetonitrile (30 mL) and reconcentrated and then in methanol
(30 mL) and reconcentrated. A solution of the residue in water
(5 mL) was applied on a column of Dowex 1 (AcO- form) (20
mL) and eluted first with water (300 mL) and then with 0.5
M acetic acid. Product-containing fractions were concentrated
in vacuo, purified by reverse phase chromatography (C-18,
water), and crystallized from H2O/acetonitrile to give white
crystals of 18 (0.136 g, 90%): mp 250-300 °C dec; UVmax 276
Diisopr opyl [2,3-Dideoxy-6-(tr iph en ylm eth yl)-â-D-er yth -
r o-h ex-2-en op yr a n osyl]p h osp h on a te (4) a n d Diisop r op yl
[2,3-Did eoxy-6-O-(t r ip h en ylm et h yl)-r-D-er yth r o-h ex-2-
en op yr a n osyl]p h osp h on a te (5). Triphenylmethyl chloride
(13.95 g, 500 mmol) and pyridine (15 mL) were added to a
solution of diol 3 (14.7 g, 50.0 mmol) in toluene (500 mL). After
stirring at room temperature for 24 h, additional toluene (500
mL) was added and the mixture was extracted sequentially
with 0.1 M hydrochloric acid, a saturated aqueous solution of
NaHCO3 (300 mL), and water (300 mL). The organic phase
was dried (MgSO4), filtered, and concentrated in vacuo to an
oil which was chromatographed on a column of silica gel using
CH2Cl2-MeOH (95:5) as eluent. 4 (â-anomer, 12.30 g, 46%)
and 5 (R-anomer, 10.45 g, 39%) were isolated as pale yellow
oils.
1
nm (ꢀ 10 532); H NMR (CDCl3) δ 3.5-3.7 (m, 2H), 4.01 (m,
1H), 4.58 (dd, J ) 18.0, 1.83 Hz, 1H), 5.29 (m, 1H), 5.96 (m,
1H), 6.18 (d, J ) 7.9 Hz, 1H), 6.53 (m, 1H), 8.26 (d, J ) 7.9
Hz, 1H); HRMS 304.0692 (M + H - glycerol), calcd for
C10H15N3O6P 304.0698. Anal. (C10H14N3O6P‚0.5H2O) C, H, N.
Diisop r op yl (4-Th ym in -1-yl-2,3,4-t r id eoxy-â-D-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (15). In a manner simi-
lar to that described for 6, 4 was coupled to N3-benzoylthy-
mine20 to furnish crude intermediate 7. 7 was deprotected first
to 11 and then to 15 following the procedures used to make
10 and 14, respectively. 15 was isolated as a white solid in
35% yield: 1H NMR (CDCl3) δ 1.3-1.4 (m, 12H), 1.90 (s, 3H),
3.3-3.5 (m, 2H), 4.02 (m, 1H), 4.3-4.6 (m, 3H), 5.9-5.6 (m,
2H), 7.62 (s, 1H), 9.02 (s, 1H).
4: 1H NMR (CDCl3) δ 1.3-1.4 (m, 12H), 3.2-3.4 (m, 2H),
4.07 (br s, 1H), 4.21 (m, 1H), 4.42 (br d, J ) 16.8 Hz, 1H),

Pyranosylphosphonic Acid Nucleotide Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1327
(4-Th ym in -1-yl-2,3,4-t r id eoxy-â-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (19). In a manner similar to that
described for 18, 15 was converted to 19 which was isolated
as white crystals in 40% yield: mp 200-250 °C dec; UVmax
270 nm (ꢀ 7925); 1H NMR (CDCl3) δ 1.87 (d, J ) 1.2 Hz, 3H),
3.46 (m, 1H), 3.67 (m, 1H), 3.99 (m, 1H), 4.55 (br d, J ) 17.7
Hz, 1H), 5.18 (m, 1H), 5.96 (m, 1H), 6.50 (m, 1H), 7.93 (d, J )
1.2 Hz, 1H). Anal. (C11H15N2O7P‚1.1H2O) C, H, N.
Diisop r op yl (4-Ad en in -9-yl-2,3,4-t r id eoxy-â-D-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (16). In a manner simi-
lar to that described for 6, 4 and N6-(phenoxyacetyl)adenine21
were coupled to form intermediate 8. 8 was directly depro-
tected first to 12 and then to 16 via the same procedures used
to make 10 and 14, respectively. 16 was obtained as a white
solid in 12% overall yield: 1H NMR (CDCl3) δ 1.3-1.5 (m,
12H), 3.82 (m, 1H), 3.95 (m, 1H), 4.2-4.3 (m, 2H), 4.6-4.9 (m,
3H), 5.32 (m, 1H), 6.2-6.5 (m, 4H), 7.78 (s, 1H), 8.32 (s, 1H).
Diisop r op yl (4-Th ym in -1-yl-2,3,4-tr id eoxy-D-th r eo-h ex-
1-en op yr a n osyl)p h osp h on a te (32). In a manner similar to
that described for 6, 5 and N3-benzoylthymine20 were reacted
to furnish crude intermediate 23 which was converted to 28
using the same procedure as described for 10. 28 was then
deprotected in a manner similar to that described for 14, to
furnish 32 in 35% overall yield from 5: 1H NMR (CDCl3) δ
1.32 (m, 12H), 1.86 (s, 3H), 2.28 (m, 1H), 2.95 (m, 1H), 3.4-
3.6 (m, 2H), 3.7 (m, 1H), 4.00 (m, 1H), 4.70 (m, 2H), 4.85 (br
d, J ) 8.1 Hz, 1H), 6.15 (d, J ) 10.2 Hz, 1H), 7.05 (s, 1H),
9.71 (br s, 1H).
(4-Th ym in -1-yl-2,3,4-t r id e oxy-D-t h r eo-h e x-1-e n op y-
r a n osyl)p h osp h on ic Acid (36). In a manner similar to that
described for 18, 32 was converted to 36. The final compound
was obtained as a white solid in 40% yield after precipitation
from H2O by acetonitrile: mp 200-250 °C dec; UVmax 272 nm
(ꢀ 2857); 1H NMR (CDCl3) δ 1.82 (d, J ) 1.2 Hz, 3H), 2.21 (m,
1H), 2.88 (m, 1H), 3.6 (m, 1H), 3.75 (m, 1H), 4.12 (m, 1H), 5.02
(m, 1H), 5.78 (m, 1H), 7.35 (d, J ) 1.2 Hz, 1H). Anal.
(C11H15N2O7P‚H2O) C, H, N.
Diisop r op yl (4-Th ym in -1-yl-2,3,4-t r id eoxy-r-D-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (42). A solution of the
crude 23 (see preparation of 32) in 10% NEt3/MeOH was kept
at room temperature for 8 h and then concentrated in vacuo.
The residue was diluted with toluene and reconcentrated to
furnish intermediate 39. The deprotection of 39 was carried
out in a manner similar to that described for 14, to afford 42
as a white solid in 29% overall yield from 5: 1H NMR (CDCl3)
δ 1.3-1.4 (m, 12H), 1.98 (s, 3H), 3.2-3.4 (m, 2H), 3.83 (m,
1H), 4.2-4.4 (m, 4H), 6.04 (m, 1H), 6.22 (m, 1H), 7.26 (s, 1H),
9.52 (s, 1H).
(4-Th ym in -1-yl-2,3,4-t r id eoxy-r-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (45). In a manner similar to that
described for 18, 42 was converted to 45. The final purification
was a precipitation from H2O by acetonitrile affording 45 as a
white solid in 35% yield: mp 200-250 °C dec; UVmax 274 nm
(ꢀ 7949); 1H NMR (CDCl3) δ 1.88 (d, J ) 1.2 Hz, 3H), 3.47 (m,
1H), 3.69 (m, 1H), 4.63 (m, 1H), 4.70 (br d, J ) 20.1 Hz, 1H),
5.18 (m, 1H), 5.96 (m, 1H), 6.53 (m, 1H), 7.46 (d, J ) 1.2 Hz,
1H). Anal. (C11H15N2O7P‚1.2H2O) C, H, N.
(4-Ad en in -9-yl-2,3,4-t r id eoxy-â-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (20). In a manner similar to that
described for 18, 16 was converted to 20 which was isolated
as white crystals in 73% yield: mp 250-300 °C dec; UVmax
1
262 nm (ꢀ 13 804); H NMR (CDCl3) δ 3.1-3.3 (m, 1H), 4.71
(d, J ) 17.2 Hz, 1H), 4.83 (m, 1H), 5.28 (m, 1H), 6.1-6.5 (m,
2H), 8.30 (s, 1H), 8.39 (s, 1H). Anal. (C11H14N5O5‚0.8H2O) C,
H, N.
Diisop r op yl (4-Gu a n in -9-yl-2,3,4-t r id eoxy-â-D-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (17). In a manner simi-
lar to that described for 6, 4 and N2-acetyl-O6-(diphenylcar-
bamoyl)guanine22 were coupled to afford crude intermediate
9 which, in a manner similar to that described for 10, was
converted to crude 13. 13 was then deprotected in a similar
fashion as for 14 to give 17 as a white solid in 51% yield from
4: 1H NMR (D2O) δ 1.4-1.5 (m, 12H), 3.1-3.4 (m, 2H), 4.58
(m, 1H), 4.84 (m, 2H), 4.9-5.1 (m, 2H), 6.2-6.4 (m, 2H), 7.76
(s, 1H).
(4-Gu a n in -9-yl-2,3,4-t r id eoxy-â-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (21). In a manner similar to that
described for 18, 17 was converted to 21 which was isolated
as white crystals in 32% yield: mp 250-300 °C dec; UVmax
1
254 nm (ꢀ 14 392); H NMR (CDCl3) δ 3.2-3.6 (m, 2H), 4.11
Diisop r op yl (4-Ad en in -9-yl-2,3,4-tr id eoxy-D-th r eo-h ex-
1-en op yr a n osyl)p h osp h on a te (33). In a manner similar to
that described for 6, 5 was reacted with 6-chloropurine to
afford crude intermediate 24 which was isolated as a foam
(∼41% yield). A solution of the foam in methanol saturated
at 0 °C with NH3 was heated at 100 °C for 8 h in a sealed
vessel. After the removal of solvent in vacuo, the residue was
chromatographed on a column of silica gel (CH2Cl2-MeOH,
95:5) to give partially purified glycal phosphonate 29 as a white
solid. In a manner similar to that described for 14, 29 was
then converted to 33 which was isolated as a white solid in
53% yield: 1H NMR (CDCl3) δ 1.2-1.4 (m, 12H), 2.55 (m, 1H),
3.03 (m, 2H), 3.69 (m, 1H), 4.18 (m, 1H), 4.73 (m, 2H), 5.24
(m, 2H), 5.89 (br s, 2H), 6.20 (m, 1H), 7.86 (s, 1H), 8.33 (s,
1H).
(br s, 1H), 4.68 (d, J ) 17.7 Hz, 1H), 5.24 (m, 1H), 6.17 (m,
1H), 6.53 (m, 1H), 8.97 (s, 1H). Anal. (C11H14N5O6P‚0.5H2O)
C, H, N.
Diisop r op yl [4-(N4-Ben zoylcytosin -1-yl)-2,3,4-tr id eoxy-
6-O-(tr iph en ylm eth yl)-r-D-th r eo-h ex-2-en opyr an osyl]ph os-
p h on a te (22). In a manner similar to that described for 6, 5
was converted to 22 which was isolated as a yellow oil in 48%
yield: 1H NMR (CDCl3) δ 1.2-1.4 (m, 12H), 3.0-3.3 (m, 2H),
3.95 (m, 1H), 4.62 (m, 1H), 4.7-4.9 (m, 2H), 5.52 (m, 1H), 6.0-
6.2 (m, 2H), 6.40 (d, J ) 7.8 Hz, 1H), 7.1-7.4 (m, 20H), 7.92
(d, J ) 7.8 Hz, 1H).
Diisop r op yl
[4-Cyt osin -1-yl-2,3,4-t r id eoxy-6-O-(t r i-
p h e n ylm e t h yl)-D-t h r eo-h e x-1-e n op yr a n osyl]p h osp h o-
n a te (27). In a manner similar to that described for 10, 22
was converted to 27 which was isolated as a white solid in
82% yield: 1H NMR (CDCl3) δ 1.3-1.4 (m, 12H), 2.1-2.2 (m,
1H), 2.7-2.8 (m, 1H), 3.2-3.4 (m, 2H), 4.21 (m, 1H), 4.6-4.9
(m, 2H), 5.41 (br d, J ) 7.2 Hz, 1H), 5.44 (d, J ) 7.3 Hz, 1H),
5.99 (m, 1H), 7.25 (m, 15H), 7.82 (d, J ) 7.3 Hz, 1H).
(4-Ad en in -9-yl-2,3,4-tr id eoxy-D-th r eo-h ex-1-en op yr a n o-
syl)p h osp h on ic Acid (37). In a manner similar to that
described for 18, 33 was converted to 37 which was isolated
as a white solid in 83% yield: mp 250-300 °C dec; UVmax 262
1
nm (ꢀ 12 481); H NMR (CDCl3) δ 2.43 (m, 1H), 3.08 (m, 1H),
Diisop r op yl (4-Cytosin -1-yl-2,3,4-tr id eoxy-D-th r eo-h ex-
1-en op yr a n osyl)p h osp h on a te (31). In a similar manner to
that described for 14, 27 was converted to 31 which was
isolated as a white solid in 83% yield: 1H NMR (CDCl3) δ 1.2-
1.4 (m, 12H), 2.23 (m, 1H), 2.93 (m, 1H), 3.33 (m, 1H), 3.73
(m, 1H), 4.04 (m, 1H), 4.5-4.7 (m, 2H), 5.36 (m, 1H), 5.74 (d,
J ) 7.5 Hz, 1H), 6.12 (m, 1H), 7.31 (d, J ) 7.5 Hz, 1H).
3.33 (m, 1H), 3.61 (m, 1H), 4.34 (m, 1H), 5.34 (m, 1H), 5.35 (d,
J ) 6.84 Hz, 1H), 5.87 (m, 1H), 8.27 (s, 1H), 8.45 (s, 1H). Anal.
(C11H14N5O5P‚1.1H2O) C, H, N.
Diisop r op yl (4-Ad en in -9-yl-2,3,4-t r id eoxy-r-D-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (43). In a manner simi-
lar to that described for 6, 5 was coupled to N6-(phenoxyacetyl)-
adenine21 to furnish intermediate 25 as an oil. A solution of
this oil in 10% NEt3/MeOH was kept at room temperature for
30 min. After dilution with toluene (100 mL), the mixture was
concentrated in vacuo. The residue was taken up in toluene
and reconcentrated to give crude 40. Treatment with 80%
aqueous acetic acid (30 mL) at 80 °C for 6 h, followed by
concentration in vacuo, left an oil which was chromatographed
on a column of silica gel (CH2Cl2-MeOH, 95:5) to furnish 43
as a white solid in 6.8% overall yield from 5: 1H NMR (CDCl3)
δ 1.3-1.4 (m, 12H), 3.2-3.5 (m, 2H), 4.10 (m, 1H), 4.54 (m,
(4-Cyt osin -1-yl-2,3,4-t r id e oxy-D-t h r eo-h e x-1-e n op y-
r a n osyl)p h osp h on ic Acid (35). In a manner similar to that
described for 18, 31 was converted to 35 which was isolated
as white crystals in 57% yield: mp 250-300 °C dec; UVmax
1
278 nm (ꢀ 10 292); H NMR (CDCl3) δ 2.12 (m, 1H), 2.84 (m,
1H), 3.5-3.7 (m, 2H), 4.02 (br s, 1H), 5.07 (d, J ) 7.3 Hz, 1H),
5.69 (d, J ) 9.5 Hz, 1H), 6.06 (d, J ) 8.0 Hz, 1H), 7.56 (d, J )
8.0 Hz, 1H); HRMS 304.0697 (M + H - glycerol), calcd for
C10H10N3O6P 306.0698. Anal. (C10H14N3O6P‚0.4H2O) C, H, N.

1328 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Alexander et al.
1H), 4.8-4.9 (m, 2H), 5.18 (m, 1H), 6.08 (br s, 2H), 6.1-6.3
(m, 2H), 8.30 (s, 1H), 8.42 (s, 1H).
1H), 5.22 (m, 1H), 5.88 (m, 1H), 6.06 (d, J ) 7.8 Hz, 1H), 6.45
(m, 1H), 8.12 (d, J ) 7.8 Hz, 1H). Anal. (C10H14N3O6P‚0.5H2O)
C, H, N.
(4-Ad en in -9-yl-2,3,4-t r id eoxy-r-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (46). In a manner similar to that
described for 18, 43 was converted to 46 which was isolated
as a crystalline solid in 58% yield: mp 250-300 °C dec; UVmax
(4-Cyt osin -1-yl-2,3,4-t r id e oxy-L -t h r eo-h e x-1-e n op y-
r a n osyl)p h osp h on ic Acid (56). In a manner similar to that
described for 18, 55 was converted to 56 which was isolated
1
1
262 nm (ꢀ 13 484); H NMR (CDCl3) δ 3.1-3.4 (m, 2H), 4.78
as a white solid in 53% yield: mp 250-300 °C dec; H NMR
(m, 1H), 4.86 (d, J ) 20.4 Hz, 1H), 5.34 (m, 1H), 6.24 (m, 1H),
6.59 (br d, J ) 10.4 Hz, 1H), 8.34 (s, 1H), 8.45 (s, 1H). Anal.
(C11H14N5O5P‚1.5H2O) C, H; N: calcd, 19.77; found, 18.59.
Diisop r op yl (4-Gu a n in -9-yl-2,3,4-tr id eoxy-D-th r eo-h ex-
1-en op yr a n osyl)p h osp h on a te (34) a n d Diisop r op yl (4-
Gu an in -9-yl-2,3,4-tr ideoxy-r-D-th r eo-h ex-2-en opyr an osyl)-
p h osp h on a te (44). In a manner similar to that described
for 6, 5 was coupled to N2-acetyl-O6-(diphenylcarbamoyl)-
guanine22 to furnish the crude intermediate 26. 26 was
converted to a mixture of 30 and 41 following the deprotection
procedure used for 10 and then deprotected further using the
procedure described for 14. The resulting mixture of 34 and
44 was separated by silica gel chromatography (CH2Cl2-
MeOH, 95:5) affording 34 in a 18% overall yield and 44 in a
12% overall yield.
(CDCl3) δ 2.14 (m, 1H), 2.91 (m, 1H), 3.53 (m, 1H), 3.72 (m,
1H), 4.08 (br s, 1H), 5.12 (d, J ) 8.1 Hz, 1H), 5.76 (m, 1H),
6.05 (d, J ) 7.5 Hz, 1H), 7.56 (d, J ) 7.5 Hz, 1H). Anal.
(C10
H14N3O6P‚0.4H2O) C, H, N.
Diisop r op yl [2,3,6-Tr id eoxy-r(a n d â)-L-er yth r o-h ex-2-
en op yr a n osyl]p h osp h on a te (58). In a manner similar to
that described for 2 and 3, 3,4-di-O-acetyl-6-deoxy-L-glucal (57)
was converted to 58 which was isolated as a yellow oil in 91%
yield: 1H NMR (CDCl3) δ 1.2-1.4 (m, 15H), 3.2-3.4 (m, 1H),
3.82 (m, 1H), 4.38 (m, 1H), 4.72 (m, 2H), 5.8-6.0 (m, 2H).
Diisopr opyl (4-Cytosin -1-yl-2,3,4,6-tetr adeoxy-â-L-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (62) a n d Diisop r op yl
(4-Cyt osin -1-yl-2,3,4,6-t et r a d eoxy-
L-th r eo-h ex-1-en op y-
r a n osyl)p h osp h on a te (66). In a manner similar to that
described for 6, 58 was converted to crude intermediate 59
which was isolated as a yellow oil (∼48% yield). The oil was
then deprotected as in the preparation of 10 to furnish a
mixture of 62 and 66 which was separated on a column of silica
34: 1H NMR (D2O) δ 1.3-1.4 (m, 12H), 2.42 (m, 1H), 3.01
(m, 1H), 3.2-3.4 (m, 2H), 4.19 (m, 1H), 4.70 (m, 2H), 5.06 (m,
1H), 6.08 (m, 1H), 7.56 (s, 1H).
44: 1H NMR (D2O) δ 1.2-1.4 (m, 12H), 3.5-3.7 (m, 3H),
4.48 (m, 1H), 4.66 (m, 2H), 5.0-5.2 (m, 2H), 5.95 (m, 1H), 6.16
(m, 1H), 6.48 (s, 2H), 7.84 (s, 1H).
(4-Gu a n in -9-yl-2,3,4-tr id eoxy-D-th r eo-h ex-1-en op yr a n -
osyl)p h osp h on ic Acid (38). In a manner similar to that
described for 18, glycal 34 was converted to 38 which was
isolated as white crystals in 96% yield: mp 250-300 °C dec;
UVmax 254 nm (ꢀ 12 500); 1H NMR (CDCl3) δ 2.4-2.5 (m, 1H),
3.0-3.1 (m, 1H), 3.4-3.7 (m, 2H), 4.32 (m, 1H), 5.26 (d, J )
6.7 Hz, 1H), 5.83 (m, 1H), 8.50 (s, 1H). Anal. (C11H14N5O6P‚
0.5H2O) C, H, N.
gel (CH2
Cl2-MeOH, 95:5). 62 and 66 were isolated as white
solids in 38% and 30% yields, respectively.
62: 1H NMR (CDCl3) δ 1.10 (d, J ) 6.3 Hz, 3H), 1.2-1.4 (m,
12H), 3,88 (m, 1H), 4.52 (d, J ) 18.3 Hz, 1H), 4.7-4.9 (m, 2H),
5.37 (br s, 1H), 5.70 (d, J ) 7.2 Hz, 1H), 5.97 (m, 1H), 6.30 (m,
1H), 7.92 (d, J ) 7.2 Hz, 1H).
66: 1H NMR (CDCl3) δ 1.21 (d, J ) 6.3 Hz, 1H), 1.3-1.5
(m, 12H), 2.14 (m, 1H), 2.80 (m, 1H), 4.13 (m, 1H), 4.6-4.8
(m, 2H), 5.23 (br d, J ) 7.3 Hz, 1H), 5.72 (d, J ) 7.5 Hz, 1H),
5.98 (m, 1H), 7.27 (d, J ) 7.5 Hz, 1H).
(4-Cytosin -1-yl-2,3,4,6-tetr adeoxy-â-L-th r eo-h ex-2-en opy-
r a n osyl)p h osp h on ic Acid (64). In a manner similar to that
described for 18, 62 was converted to 64 which was isolated
as white crystals in 56% yield: mp 250-300 °C dec; UVmax
276 nm (ꢀ 10 735); 1H NMR (CDCl3) δ 1.03 (d, J ) 6.5 Hz,
3H), 4.03 (m, 1H), 4.50 (d, J ) 17.6 Hz, 1H), 5.12 (m, 1H),
5.94 (m, 1H), 6.04 (d, J ) 7.3 Hz, 1H), 6.43 (m, 1H), 8.05 (d, J
) 7.3 Hz, 1H); HRMS 288.0756 (M + H - glycerol), calcd for
C10H15N3O5P 288.0749. Anal. (C10H14N3O5P‚H2O) C, H; N:
calcd, 12.99; found, 13.77.
(4-Gu a n in e-9-yl-2,3,4-tr id eoxy-r-D-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on ic Acid (47). In a manner similar to that
described for 18, 44 was converted to 47 which was isolated
as white crystals in 86% yield: mp 250-300 °C dec; UVmax
1
254 nm (ꢀ 13 805); H NMR (CDCl3) δ 3.2-3.5 (m, 2H), 4.74
(br s, 1H), 4.82 (d, J ) 20.7 Hz, 1H), 5.16 (br s, 1H), 6.2 (m,
1H), 6.58 (d, J ) 9.7 Hz, 1H), 8.26 (s, 1H). Anal. (C11H14N5O6P‚
0.5H2O) C, H, N.
Diisop r op yl [2,3-Did eoxy-6-O-(tr ip h en ylm eth yl)-â(a n d
r)-L-er yth r o-h ex-2-en op yr a n osyl]p h osp h on a te (49). In a
manner similar to that described for 2-4 (and 5) tri-O-acetyl-
L-glucal (48) was converted to 49 which was isolated as a pale
yellow oil in 94% overall yield: 1H NMR (CDCl3) δ 1.3-1.4
(m, 12H), 3.2-3.3 (m, 2H), 4.07 (m, 1H), 4.21 (m, 1H), 4.2-
4.4 (m, 1H), 4.7-4.9 (m, 2H), 5.8-6.0 (m, 2H), 7.2-7.5 (m,
15H).
(4-Cytosin -1-yl-2,3,4,6-tetr a d eoxy-L-th r eo-h ex-1-en op y-
r a n osyl)p h osp h on ic Acid (69). In a manner similar to that
described for 18, glycal 66 was converted to 69 which was
isolated as white crystals in 87% yield: mp 250-300 °C dec;
1
UVmax 276 nm (ꢀ 10 323); H NMR (CDCl3) δ 1.16 (d, J ) 6.3
Hz, 3H), 2.20 (m, 1H), 2.88 (m, 1H), 4.21 (m, 1H), 5.04 (d, J )
7.8 Hz, 1H), 5.76 (m, 1H), 6.06 (d, J ) 7.8 Hz, 1H), 7.54 (d, J
) 7.8 Hz, 1H); HRMS 288.0754 (M + H - glycerol), calcd for
C10H15N3O5P 288.0749. Anal. (C10H14N3O5P‚H2O) C, H, N.
Diisop r op yl (4-Cyt osin -1-yl-2,3,4-t r id eoxy-â-L-th r eo-
h ex-2-en op yr a n osyl)p h osp h on a te (52) a n d Diisop r op yl
(4-Cytosin -1-yl-2,3,4-tr ideoxy-L-th r eo-h ex-1-en opyr an osyl)-
p h osp h on a te (55). In a manner similar to that described
for 6, 49 was converted to crude 50 which was isolated as a
yellow oil (48% yield). The oil was treated with NH3/MeOH
as described for 10, to afford a mixture of partially deprotected
intermediates 51 and 54 which was deprotected further using
acetic acid as was done to prepare 14. The resulting mixture
of 52 and 55 was separated by silica gel chromatography (CH2-
Cl2-MeOH, 95:5) to furnish each as a white solid.
Diisop r op yl
(4-Th ym in -1-yl-2,3,4,6-t et r a d eoxy-â-L-
th r eo-h ex-2-en op yr a n osyl)p h osp h on a te (63) a n d Diiso-
p r op yl (4-Th ym in -1-yl-2,3,4,6-tetr a d eoxy-L-th r eo-h ex-1-
en op yr a n osyl)p h osp h on a te (67). In a manner similar to
that described for 6, the anomeric mixture 58 was converted
to crude intermediate 60 which was isolated as a yellow oil.
This mixture of anomers was then treated with NH3/MeOH
as described for 10 to furnish a mixture of 63 and 67.
Chromatographic separation on silica gel (CH2Cl2-MeOH, 95:
5) afforded 63 as a white solid in 20% yield and 67 as a white
solid in 19% yield.
52: 32% yield from 50; 1H NMR (CDCl3) δ 1.2-1.4 (m, 12H),
3.2-3.6 (m, 2H), 3.90 (m, 1H), 4.57 (d, J ) 18 Hz, 1H), 4.6-
4,9 (m, 2H), 5.41 (m, 1H), 5.78 (d, J ) 7.2 Hz, 1H), 6.05 (m,
1H), 6.32 (m, 1H), 8.00 (d, J ) 7.2 Hz, 1H).
63: 1H NMR (CDCl3) δ 1.13 (d, J ) 5.4 Hz, 3H), 1.3-1.5
(m, 12H), 1.95 (s, 3H), 3.88 (m, 1H), 4.53 (d, J ) 18.9 Hz, 1H),
4.80 (m, 2H), 5.09 (m, 1H), 5.95 (m, 1H), 6.33 (m, 1H), 7.80 (s,
1H), 8.51 (s, 1H).
55: 53% yield from 50; 1H NMR (CDCl3) δ 1.2-1.3 (m, 12H),
2.25 (m, 1H), 2.98 (m, 1H), 3.36 (m, 1H), 3.40 (m, 1H), 4.06
(m, 1H), 4.5-4.7 (m, 2H), 5.39 (m, 1H), 5.76 (d, J ) 7.4 Hz,
1H), 6.12 (m, 1H), 7.32 (d, J ) 7.4 Hz, 1H).
67: 1H NMR (CDCl3) δ 1.27 (d, J ) 5.3 Hz, 3H), 1.3-1.4
(m, 12H), 1.88 (s, 3H), 2.18 (m, 1H), 2.85 (m, 1H), 4.11 (m,
1H), 4.74 (m, 2H), 5.04 (d, J ) 7.8 Hz, 1H), 6.08 (m, 1H), 7.08
(s, 1H), 8.82 (s, 1H).
(4-Cyt osin -1-yl-2,3,4-t r id eoxy-â-L-th r eo-h ex-2-en op y-
r a n osyl)p h osp h on a te (53). In a manner similar to that
described for 18, 52 was converted to 53 which was isolated
as white crystals in 83% yield: mp 250-300 °C dec; 1H NMR
(CDCl3) δ 3.4-3.6 (m, 2H), 3.93 (m, 1H), 4.50 (d, J ) 18.1 Hz,
(4-Th ym in -1-yl-2,3,4,6-tetr adeoxy-â-L-th r eo-h ex-2-en opy-
r a n osyl)p h osp h on ic Acid Sod iu m Sa lt (65). In a manner
similar to that described for 18, 63 was converted to 65. After

Pyranosylphosphonic Acid Nucleotide Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1329
drug as controls. After 7-10 days at 37 °C, the wells were
reverse phase chromatography, the product was dissolved in
H2O and the pH adjusted to 7 by the addition of a saturated
aqueous solution of sodium perchlorate. After concentrating
in vacuo to 2 mL, the product was precipitated by the addition
of acetone. Isolation of the solid afforded pure 65 in 73%
stained with crystal violet and scored visually under
a
microscope. All inhibitors were assayed for anti-HCMV activ-
ity in duplicate. The data were plotted as percent inhibition
of the average plaque number vs the concentration of drug.
The IC50 value of the inhibitor is defined as the concentration
of drug that decreased the number of plaques to 50% of the
number present in the control wells with no drug.
The drugs’ cytotoxic effect was assessed by incubating
uninfected NHDF monolayers with serial dilutions of drug at
37 °C, 4% CO2, for 7-10 days and then washing with sterile
PBS, adding XTT medium, incubating at 37 °C for 45 min,
and then reading absorbance at 450 nm.24,25
HSV-2 Assa y.24,25 Serial dilutions of drug in Earl’s MEM
with 10% FBS were added to wells of a 96-well plate containing
MRC-5 cells. After incubation at 37 °C, 5% CO2, for 15 min,
∼100 PFU/well HSV-2 (E 194) was added to the wells and
incubated at 37 °C, 5% CO2, for 2.5-3 days until ca. 90%
killing was present in the control (no drug) wells. The medium
was then removed, the cells were washed with sterile PBS,
and XTT medium was added. After incubating at 37 °C for
30-40 min, absorbance was read at 450-650 nm.
1
yield: mp 200-250 °C dec; UVmax 274 nm (ꢀ 9973); H NMR
(CDCl3) δ 1.06 (d, J ) 5.86 Hz, 3H), 1.87 (s, 3H), 3.99 (m, 1H),
4.41 (m, 1H), 4.99 (m, 1H), 5.86 (m, 1H), 6.48 (m, 1H), 7.38 (s,
1H); HRMS 325.0578 (M + H - glycerol), calcd for C11H15N2O6-
NaP 325.0565. Anal. (C11H14N2O6PNa‚0.4H2O) C, H, N.
(4-Th ym in -1-yl-2,3,4,6-tetr a d eoxy-L-th r eo-h ex-1-en op y-
r a n osyl)p h osp h on ic Acid (70). In a manner similar to that
described for 18, 67 was converted to 70. After reverse phase
chromatography, the product was dissolved in H2O and the
pH was adjusted to 7 by the addition of a saturated aqueous
solution of sodium perchlorate. After concentrating in vacuo
to 2 mL, the product was precipitated by the addition of
acetone. Isolating the solid afforded pure 70 in 80% yield: mp
1
200-250 °C dec; UVmax 274 nm (ꢀ 8306); H NMR (CDCl3) δ
1.17 (d, J ) 1.17 Hz, 3H), 1.88 (d, J ) 1.0 Hz, 3H), 2.16 (m,
1H), 2.83 (m, 1H), 4.16 (m, 1H), 4.91 (m, 1H), 5.61 (m, 1H),
7.42 (d, J ) 1.0 Hz, 1H); HRMS 325.0573 (M + H - glycerol),
calcd for
C11H15N2O6NaP 325.0578. Anal. (C11H14N2O6-
PNa‚0.5H2O) C, H, N.
Ack n ow led gm en t . Thanks are due to David
Barkhimer and Andrew Mulato for biological evalua-
tions, J ulie Cherrington for manuscript review, and
Melanie Macke for expert manuscript preparation.
Diisop r op yl (4-Ad en in -9-yl-2,3,4,6-tetr a d eoxy-L-th r eo-
h ex-1-en op yr a n osyl)p h osp h on a te (68). In a manner simi-
lar to that for 6, the anomeric mixture 58 was reacted with
6-chloropurine to afford intermediate 61 which was isolated
as a yellow oil (∼34% yield). Using NH3/MeOH as described
in the preparation of 29, the oil was converted to 68 which
was isolated as a white solid in 18% yield from 58: 1H NMR
(CDCl3) δ 1.13 (d, J ) 6.9 Hz, 3H), 1.3-1.4 (m, 12H), 2.37 (m,
1H), 2.94 (m, 1H), 4.34 (m, 1H), 4.77 (m, 2H), 5.02 (br d, J )
6.9 Hz, 1H), 5.72 (br d, 2H), 6.06 (m, 1H), 7.98 (s, 1H), 8.35 (s,
1H).
Refer en ces
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New York, 1970; pp 110-217.
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Nucleotides 1994, 13, 1271-1295. (b) Nucleosides and Nucletides
as Antitumor and Antiviral Agents; Chu, C. K., Baker, D. C.,
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(4-Ad en in -9-yl-2,3,6-tr id eoxy-L-th r eo-h ex-1-en op yr a n o-
syl)p h osp h on ic Acid (71). In a manner similar to that
described for 18, 71 was prepared from glycal 68 and isolated
as white crystals in 88% yield: mp 172 °C; UVmax 262 nm (ꢀ
12 808); 1H NMR (CDCl3) δ 1.01 (d, J ) 6.4 Hz, 3H), 2.37 (m,
1H), 2.99 (m, 1H), 4.37 (m, 1H), 4,99 (d, J ) 6.8 Hz, 1H), 5.78
(m, 1H), 8.10 (s, 1H), 8.24 (s, 1H); HRMS 312.0862 (M + H -
glycerol), calcd for C11H15N5O4P 312.0861. Anal. (C11H14N5O4P‚
0.4H2O) C, H, N.
(3) (a) Verheggen, I.; Van Aerschot, A.; Toppet, S.; Snoeck, R.;
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Diisop r op yl (4-Cyt osin -1-yl-2,3,4-t r id eoxy-â-D-th r eo-
h exop yr a n osyl)p h osp h on a te (72). A solution of 14 (0.195
g, 0.5 mmol) in methanol (10 mL) was stirred under H2 at 1
atm for 4 h in the presence of 10% Pd on carbon (0.05 g). The
mixture was filtrated and concentrated in vacuo to give
phosphonate 72 as a white solid (0.190 g, 98%): 1H NMR
(CDCl3) δ 1.2-1.4 (m, 12H), 1.7-2.1 (m, 4H), 3.49 (m, 1H),
3.67 (m, 1H), 3.88 (m, 2H), 4.76 (m, 2H), 4.97 (m, 1H), 5.71 (d,
J ) 7.8 Hz, 1H), 8.14 (d, J ) 7.8 Hz, 1H).
(4-Cytosin -1-yl-2,3,4-tr ideoxy-â-D-th r eo-h exopyr an osyl)-
p h osp h on ic Acid (73). A solution of 72 (0.195 g, 0.49 mmol),
2,4-lutidine (0.6 mL, 5 mmol), and bromotrimethylsilane (0.66
mL, 5 mmol) in acetonitrile (5 mL) was stirred at room
temperature for 24 h. The solvent was removed in vacuo, and
the residue was taken in methanol (10 mL), reconcentrated,
and then dissolved in water (5 mL) and applied on a Dowex 1
(AcO- form) column (20 mL). After eluting with water (300
mL) followed by 0.1 M acetic acid, product-containing fractions
were evaporated and the residue was precipitated from H2O/
acetonitrile to give 73 as a white solid (0.136 g, 90%): mp 250-
1
300 °C dec; UVmax 276 nm (ꢀ 9503); H NMR (CDCl3) δ 1.7-
1.9 (m, 2H), 2.0-2.2 (m, 2H), 3.5-3.7 (m, 2H), 3.82 (m, 1H),
3.91 (m, 1H), 4.71 (m, 1H), 6.05 (d, J ) 8.0 Hz, 1H), 8.51 (d, J
) 8.0 Hz, 1H); HRMS 306.0859 (M + H - glycerol), calcd for
C
10H17N3O6P 306.0855. Anal. (C10H16N3O6P‚1.2H2O) C, H, N.
Biologica l Meth od s: HCMV Assa y.23 Confluent mono-
layers of normal human dermal fibroblasts (NHDF) in a 24-
well plate were infected with HCMV (Towne). After 1-2 h at
37 °C, the medium was replaced and serial dilutions of the
drug were applied to the wells. Some wells were left free of

1330 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
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