A 4′-C-ethynyl-2′,3′-dideoxynucleoside analogue highlights the role of the 3′-OH in anti-HIV active 4′-C-ethynyl- 2′-deoxy nucleosides

Article abstract of DOI:10.1021/jm049550o

4′-C-Ethynyl-2′-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several

Full text of DOI:10.1021/jm049550o

J . Med. Chem. 2004, 47, 5041-5048
5041
A 4′-C-Eth yn yl-2′,3′-Did eoxyn u cleosid e An a logu e High ligh ts th e Role of th e
3′-OH in An ti-HIV Active 4′-C-Eth yn yl-2′-d eoxy Nu cleosid es
Maqbool A. Siddiqui,† Stephen H. Hughes,‡ Paul L. Boyer,‡ Hiroaki Mitsuya,§ Que N. Van,| Clifford George,
Stefan G. Sarafinanos,O and Victor E. Marquez*,†
Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, NIH, Frederick, Maryland 21702,
HIV Drug Resistance Program, Center for Cancer Research, NCI-Frederick, NIH, Frederick, Maryland 21702,
Experimental Retrovirology Section, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, NMR Group,
Laboratory of Proteomics and Analytical Technologies, SAIC Frederick, Frederick, Maryland 21702, Laboratory for the
Structure of Matter, Naval Research Laboratory, Washington, D.C. 20375, and Center for Advanced Biotechnology and
Medicine and Chemistry Department, Rutgers University, Piscataway, New J ersey 08854
Received J une 8, 2004
4′-C-Ethynyl-2′-deoxynucleosides belong to a novel class of nucleoside analogues endowed with
potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones.
Although favorable selectivity indices were reported for several of these analogues, some concern
still exists regarding the 3′-OH group and its role in cellular toxicity. To address this problem,
we removed the 3′-OH group from 4′-C-ethynyl-2′-deoxycytidine (1a ). This compound was chosen
because of its combined high potency and low selectivity index. The removal of the 3′-OH was
not straightforward; it required a different synthetic approach from the one used to synthesize
the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4′-C-ethynyl-
2′,3′-dideoxycytidine analogue (r a c-1h ) was obtained after 13 steps. In a cellular assay, r a c-
1h was completely inactive (0.001-10 µM) against HIVLAI, demonstrating the critical importance
of the 3′-OH for antiviral activity. To determine whether the role of the 3′-OH was essential
for the phosphorylation of the compound by cellular kinases or for inhibition of DNA
polymerization, we synthesized and tested the 5′-triphosphate (r a c-1h -TP ) for its ability to
inhibit HIV reverse transcriptase (RT). r a c-1h -TP was slightly more potent than AZT-5′-
triphosphate against wild-type HIV RT, suggesting that the role of the 3′-OH is crucial only
for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of r a c-1h
into en t-1h (â-D-dideoxyribo) and en t-14 (â-L-dideoxyribo) and the synthesis of the corresponding
5′-triphosphates established the stereochemical assignment based on HIV RT’s preference for
the â-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant
with a marked preference for incorporating nucleosides in the D-configuration.
In tr od u ction
4′-C-Ethynyl-2′-deoxy-â-D-nucleosides (1a -e) have
been identified as some of the most potent analogues
against HIV-1, including several multidrug-resistant
strains and HIV-2.1,2 Although no studies with the
corresponding 5′-triphosphates have been performed to
elucidate the exact mechanism of antiretroviral activity,
the antiviral profile fits that of an NRTI with a mech-
anism of action similar to that of AZT. This means that
4′-C-ethynyl nucleosides suppress HIV replication at or
around the step of reverse transcription. Indeed, the fact
that the anti-HIV activity of the cytidine (1a ) and
guanosine (1e) analogues was reversed by the addition
of their physiologic 2′-deoxynucleoside counterparts
Previous reports of other 4′-substituted-2′-deoxy-â-D-
nucleosides, such as 4′-azidothymidine (1f), have shown
that following intracellular anabolism to the 5′-tri-
phosphate, HIV reverse transcriptase (RT) efficiently
incorporated the nucleotide, which prevented further
chain elongation of the viral DNA.3,4 Because these
compounds bear no structural resemblance to tradi-
tional chain terminators, all of which lack the 3′-OH
group, they must have a different mechanism of action.
In the case of 4′-azidothymidine (1f), the rate of incor-
poration of the 5′-triphosphate by cellular polymerases
provides strong evidence that 4′-C-ethynyl-2′-deoxy-â-
D-nucleosides serve as substrates for HIV RT.2
* Author to whom correspondence should be addressed. E-mail:
marquezv@dc37a.nci.nih.gov.
† Laboratory of Medicinal Chemistry, NCI-Frederick.
‡ HIV Drug Resistance Program, NCI-Frederick.
§ Experimental Retrovirology Section, NCI, NIH.
| NMR Group, Laboratory of Proteomics and Analytical Technolo-
gies, SAIC Frederick.
Laboratory for the Structure of Matter, Naval Research Labora-
tory.
O Rutgers University.
10.1021/jm049550o CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/01/2004

5042 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Siddiqui et al.
Sch em e 1a
was found to be dramatically different when compared
to that of HIV RT. Although the rate of incorporation
for the former was quite low, HIV RT was able to
incorporate two consecutive molecules efficiently. The
subsequent distortion of the growing primer brought
about by this incorporation seems to prevent further
DNA chain elongation, thus causing a delayed chain
termination.3,4 Other structurally similar compounds
probably have comparable mechanisms of action, for
example, 4′-cyanothymidine (1g) and the corresponding
cytidine and uridine analogues, all of which are potent
inhibitors of HIV replication in cell culture.5 As a group,
4′-substituted-2′-deoxy-â-D-nucleosides are quite attrac-
tive because of the intriguing connection between their
novel mechanism of action and their potent activity
against multidrug-resistant (MDR) HIV-1 strains. Their
efficacy against MDR strains may depend on two
important factors: (1) delayed chain termination occur-
ring beyond the polymerase active site and (2) difficul-
ties in selecting mutants that discriminate against the
incorporation of analogues that still retain the natural
3′-OH.2
Extensive studies with other 4′-substituted-2′-deoxy-
â-D-nucleosides with a thymine nucleobase and a variety
of substituents, including ethyl, hydroxyethyl, and
ethenyl, at the 4′-position demonstrated the superior
potency of the ethynyl group.6 Similarly, for compounds
with a cytosine nucleobase, the ethynyl group as in 1a
was the most potent analogue although some host
cellular toxicity was encountered.7 Surprisingly, the
5-fluorocytosine analogue (1b) was less toxic and quite
potent. Other important structural changes including
the use of purine nucleobases, such as adenine (1c) and
2,6-diaminopurine (1d ), produced compounds active
against MDR HIV-1 that were less toxic than their
counterparts with pyrimidine nucleobases.1
Because the 3′-OH in all of these 4′-substituted
nucleosides could function as a key element for sub-
strate recognition by cellular kinases and/or poly-
merases, we decided to investigate the importance of
the 3′-OH by deleting it from the most attractive series
of 4′-substituted nucleosides, the 4′-C-ethynyl-2′-deoxy-
â-D-nucleosides. Previous studies have shown that
removal of the 3′-OH from the 4′-azido (1f) and 4′-cyano
(1g) analogues resulted either in a complete loss of
activity or a significant reduction in potency.5,8 However,
with the structurally related 3′,4′-fused oxetane deriva-
tive with a thymine nucleobase, there was only a
moderate drop in potency, suggesting that the ether
oxygen could have partially fulfilled the role of the 3′-
OH.9 Despite the expected loss of activity similar to
what was seen with the 4′-azido analogue (1f), removing
the 3′-OH from 4′-C-ethynyl-2′-deoxy-â-D-nucleosides to
generate a 4′-C-ethynyl-2′,3′-dideoxy-â-D-nucleoside, such
as 1h , provided the opportunity to assess the role of the
3′-OH during activation and polymerization and in
generating cellular toxicity. Because the issue of toxicity
was very important, cytosine analogue 1h was selected
to be the target because its progenitor 1a combined
potent activity with significant cellular toxicity.
a
(a) (CH3CO)2O/pyridine (86%); (b) TMSOTf, CH2Cl2 (100%);
(c) BCl3, CH2Cl2, -78 °C (r a c-6, 49%; r a c-5, 25%).
in which compound 2 was generated only as an inter-
mediate and not fully characterized.10 The procedure
was repeated, and compound 2 was completely charac-
terized. Acylation of the free hydroxyl group to give 3
followed by standard nucleoside synthesis with per-
silylated N4-benzoylcytosine proceeded uneventfully to
afford an inseparable mixture of two diastereoisomers
(4, Scheme 1). The removal of the benzyl protecting
group gave a separable mixture of r a c-5 and r a c-6,
which differ in terms of their relative stereochemistry.
Because the correct stereochemical assignment was not
clarified until the target compounds were reached (vide
infra), the same chemical steps were performed sepa-
rately on r a c-5 and r a c-6 but are shown only for one
diastereoisomer (r a c-5, Scheme 2).
The oxidation of the hydroxymethyl group to a formyl
group (r a c-7) was followed by Wittig olefination with
(chloromethyl)triphenylphosphonium chloride to afford
chlorovinyl derivative r a c-8. Without isolation, the
chlorovinyl group was converted to the ethynyl group
after treatment with n-butyllithium in tetrahydrofuran
to give r a c-9. Finally, deprotection of the p-methoxy-
phenyl group with ammonium cerium (IV) nitrate
provided the desired target r a c-1h . Following an identi-
cal approach from r a c-6 produced r a c-10. At this stage,
NMR nOe buildup data adequately determined the
relative stereochemistry. In r a c-10, the nOe build ups
indicated a distance between H-5′a,b and H-1′ (nucleo-
side numbering) of 3.3 Å, whereas no nOe was observed
between the same set of protons in r a c-1h . Buildup
Ch em istr y
The required 4-branched-2,3-dideoxysugar 2 was
prepared according to a previously published method

4′-C-Ethynyl-2′,3′-Dideoxynucleoside Analogue
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5043
Sch em e 2a
Sch em e 3a
a
(a) DCC, DMSO, 0 °C (93%); (b) ClCH2P(C6H5)3Cl, n-BuLi,
THF, -78 °C; (c) n-BuLi, THF, -78 °C (48.5% two steps); (d)
(NH4)2Ce(NO3)6, CH3CN:H2O, 0 °C (83%).
a
(a) i. TMSCl, pyridine, ii. PhCOCl, iii. NH4OH (68.5%); (b)
lipase PS-C Amano I, CH2dCHOAc, CCl4/CHCl3 (en t-13,
58%; en t-12, 40.5%) (c) satd NH3/CH3OH (70%); (d) per-
formed by TriLink Biotechnologies, Inc., San Diego, CA (www.
trilinkbiotech.com).
acetate, the less polar monoacetate (en t-13) was ob-
tained as a single enantiomer (91% ee) after flash
column chromatography. The mixture of unreactive
alcohols was then treated for a second time with the
enzyme under the same conditions to give, after column
chromatography, enantiomerically pure en t-12 (98% ee).
The removal of the N4-benzoyl group from en t-12 and
the acetyl/N4-benzoyl groups from en t-13 gave
the desired â-D-dideoxyribo targets: en t-1h and â-L-
dideoxyribo enantiomer en t-14. This assignment was
based on the capacity of HIV-1 RT to discriminate
between the enantiomers (vide infra) and, more re-
cently, on the synthesis of the authentic â-D-dideoxyribo
enantiomer (en t-1h ) obtained from a chiral lactone
precursor to 212 using a method similar to that shown
in Schemes 1 and 2. The synthesis of the corresponding
5′-triphosphates, en t-1h -TP (â-D-dideoxyribo) and en t-
14-TP (â-L-dideoxyribo), was performed by conventional
methods. The 5′-triphosphate of the racemate, r a c-1h -
TP , was also synthesized as a reference.
F igu r e 1. Displacement elliposoid plot of r a c-1h drawn at
the 30% probability level.
curves were obtained by irradiating the key target
protons on both samples and using an internuclear
distance of 2.4 Å between H-6 and H-5 (nucleoside
numbering) as a reference for the calculations. During
the NMR experiments, it was noticed that r a c-1h
crystallized readily and provided suitable crystals for
X-ray analysis. As shown in Figure 1, the crystal
structure of r a c-1h corresponded to the desired (2R,5R
or 2S,5S)-(()-1-(2′,3′-dideoxy-4′-C-ethynyl-ribopento-
furanosyl)cytosine.
Biologica l Activity
At this point, a lipase-catalyzed approach was used
to resolve r a c-1h . To avoid complications with possible
interference by the exocyclic amino group during trans
esterification with vinyl acetate, which included acety-
lation or formation of a Schiff base with the released
acetaldehyde, this group was protected as a benzoyl
amide using the well-known transient protection method
to give r a c-11 (Scheme 3).11 Following a 10-day incuba-
tion of r a c-11 with lipase PS-C “Amano” and vinyl
Relative to 4′-C-ethynyl-2′-deoxycytidine (1a ), dideoxy
analogue r a c-1h was inactive in vitro against HIVLAI
when tested in a concentration range between 0.001 and
10 µM in MT-2 and MT-4 cells. Under the same
experimental conditions, control AZT had an IC50 of
0.014 µM. These results underscore the critical role of
the 3′-OH group for anti-HIV activity in a cellular
system. To determine whether the role of the 3′-OH was
crucial for the activation step(s) or for the inhibition of

5044 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Siddiqui et al.
F igu r e 3. Superposition of en t-1h -TP and en t-14-TP at the
active site of HIV RT showing a measurable deviation of the
4′-ethynyl moiety.
2, 2 versus 1). On the basis of these results, it would
appear that the 3′-OH of the â-D enantiomer of 1a is
critical for phosphorylation by cellular kinases, but in
the L-enantiomer, it must play a negative role by
occupying an unfavorable position in the active site of
the kinase. The removal of the 3′-OH from either
enantiomer produces inactive compounds in cell-based
assays, but to a varying degree, both 5′-triphosphates
seem to be acceptable substrates for HIV-1 RT. Accord-
ing to a recent report, the combined removal of the 3′-
OH and the flattening of the dideoxyribose ring by a
2′,3′ double bond restores anti-HIV activity in MT-2
cells.20 This finding seems to be consistent with our own
observations correlating a reduced puckering amplitude
with substrate recognition by both cellular kinases and
polymerases.21
F igu r e 2. Inhibition of HIV RT wild type and M184V mutant
strains by racemic (r a c-1h TP ) and enantiomeric (en t-1h TP
and en t-14TP ) 5′-triphosphates of 1-(2′,3′-dideoxy-4-C-ethynyl-
ribofuranosyl)cytosine: r a c-1h -TP (wild type ) b); en t-1h -
TP (wild type ) [); en t-1h -TP (M184V ) 1); en t-14-TP (wild
type ) 2); en t-14-TP (M184V ) 9).
HIV-1 viral DNA polymerization, we synthesized and
tested the 5′-triphosphate (r a c-1h -TP ) for its ability to
inhibit HIV RT. Against wild-type HIV-1 RT, using a
single-stranded M13mp18 DNA template and a -47
sequencing primer, r a c-1h -TP was slightly more potent
than AZT-5′-triphosphate (not shown), suggesting that
the 3′-OH plays a critical role in the activation of the
drug by cellular kinases (Figure 2, b). We expected the
enzyme to discriminate between enantiomers en t-1h -
TP (â-D-dideoxyribo) and en t-14-TP (â-L-dideoxyribo)
on the basis of calculations from the molecular docking
of the 5′-triphosphates into the active site of HIV RT
(vide infra), which predicted that the â-D-dideoxyribo
enantiomer (en t-1h -TP ) would be the more active
isomer. Indeed, the enantiomer presumed to be the â-D-
dideoxyribo enantiomer (en t-1h -TP ) was more potent
in blocking DNA synthesis by wild-type HIV-1 RT than
its antipode en t-14-TP (Figure 2, [ versus 2). The fact
that the M184V mutant of HIV-1 RT has a greater
selectivity against â-L-nucleosides than does wild
type13-16 also confirmed the assignment of D- and
D-enantiomers: the â-L-dideoxyribo enantiomer (en t-14-
TP ) was virtually inactive (Figure 2, 9), whereas the
â-D-dideoxyribo enantiomer (en t-1h -TP ) strongly in-
hibited the M184 mutant RT despite a low level of
resistance (Figure 2, 1). This low level of resistance of
M184V for the â-D-dideoxyribo (en t-1h -TP ) enantiomer
is not surprising because M184V has been reported to
show a low level of resistance to ddCTP.17,18
Cr ysta l Str u ctu r e a n d Dock in g of
5′-Tr ip h osp h a tes a t th e Active Site of HIV RT
On the basis of the torsion angles obtained from the
crystal structure (Figure 1), the dideoxyribose moiety
of r a c-1h has a P value of 43.49° in the pseudorotational
cycle, almost exactly in the middle of a 3T4 (P ) 36°)
and 4′-exo (4E, P ) 54°) conformations. This is clearly
in the Northern Hemisphere but slightly beyond the
range that is observed for most conventional nucleosides
(0-36°). The molecule also has a highly puckered ring
with a νmax of 37.10°. The X-ray coordinates of r a c-1h
were used to construct the 5′-triphosphates of en t-1h -
TP and en t-14-TP for computer docking studies with
HIV RT. When the two structures are superposed at the
polymerase site of an HIV RT/DNA/nucleotide ternary
complex, there is a measurable deviation in the direction
of the 4′-ethynyl moiety between the two enantiomers
(Figure 3). The D enantiomer (en t-1h -TP ) appears to
fit nicely at the active site of wild-type HIV RT (Figure
4A), and consistent with the low level of resistance
observed against the M184V mutant (Figure 2, 1), the
docking in Figure 4C shows some negative steric
interaction with Val184. The situation with the
L
enantiomer (en t-14-TP ) would indicate that relative to
en t-1h -TP the fit with respect to Met184 is somewhat
tighter (Figure 4B), plus there could be some steric
interactions in wild-type HIV RT with Tyr115 and/or
Ala114, or with both (Figure 5). It is possible that the
steric interactions of the L enantiomer with these amino
acids could be alleviated either by a torsional rotation
In light of a recent investigation reporting that the
L-enantiomer of 1a is inactive against the HIV virus in
cell culture, 19 it is interesting that en t-14-TP is active
against wild-type HIV-1 RT. In fact, the level of potency
of en t-14-TP against wild-type HIV-1 RT is identical
to that of en t-1h -TP against the M184V mutant (Figure

4′-C-Ethynyl-2′,3′-Dideoxynucleoside Analogue
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5045
F igu r e 4. (A) â-D-enantiomer (en t-1h -TP ) at the active site of wild-type HIV RT. Volumes for the 4′-ethynyl group (green) and
methionine184 (yellow). (B) â-L-enantiomer (en t-14-TP ) at the active site of wild-type HIV RT. Volumes for the 4′-ethynyl group
(yellow) and methionine184 (green). (C) â-D-enantiomer (en t-1h -TP ) at the active site of M184V HIV RT. Volumes for the 4′-
ethynyl group (green) and methionine184 (yellow). (D) â-L-enantiomer (en t-14-TP ) at the active site of M184V HIV RT. Volumes
for the 4′-ethynyl group (yellow), methionine184 (green), and steric clash (orange).
F igu r e 5. â-L-enantiomer (en t-14-TP ) at the active site of wild-type HIV RT. Volumes for the 4′-ethynyl group (green), alanine
and tyrosine (yellow), and steric clash (orange).
of Tyr115 or a small rearrangement of the L nucleotide,
explaining the lower level of potency observed for this
enantiomer against wild-type HIV-1 RT (Figure 2, 2).
However, it is clear that for the M184V mutant the
steric clash between the Cγ of the â-branched valine
with the 4′-ethynyl group of en t-14-TP (Figure 4D) and
possibly with the L-dideoxy ring (not shown) would
prevent the L enantiomer from binding to the active site
in a productive manner, explaining its lack of activity
(Figure 2, 9).
for other 4′-substituted 2′-deoxynucleosides that are
active against HIV (vide supra). The potent inhibition
of HIV-1 RT by the 5′-triphosphate (r a c-1h -TP ) con-
firms that the failure of this agent to exert good anti-
HIV activity in the cell culture results from the failure
of the cellular kinases to generate the requisite 5′-
triphosphate. In the present work, the racemate 1-(2′,3′-
dideoxy-4′-C-ethynyl-ribopentofuranosyl)cytosine (r a c-
1h ) was resolved into its components, â-D-dideoxyribo
(en t-1h ) and â-L-dideoxyribo (en t-14), by a lipase-
catalyzed reaction. The assignment of the absolute
stereochemistry was confirmed by HIV RT’s preference
for the natural â-D-configuration. This assignment was
confirmed using the M184V mutant, which has a much
greater preference for the D enantiomers. The synthesis
of authentic en t-1h also confirmed this assignment
because its 5′-triphosphate showed identical activity as
Discu ssion
The inactivity of 1-(2′,3′-dideoxy-4′-C-ethynyl-ribo-
pentofuranosyl)cytosine (r a c-1h ) as an inhibitor of HIV
replication in cultured cells highlights the importance
of the 3′-OH as a key determinant for activity for the
4′-C-ethynyl-2′-deoxy-â-D-nucleosides and, by extension,

5046 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Siddiqui et al.
obtained was filtered, and the organic layer was separated,
dried (MgSO4), and concentrated under vacuum. The crude
product that was obtained was purified by flash chromatog-
raphy on silica gel using EtOAc/hexanes (1:1 f pure EtOAc)
to give a quantitative yield of a yellowish foam consisting of
an inseparable mixture of two diastereoisomers (4). This
material was carried out as a mixture in the following
debenzylation step.
(2R,5R or 2S,5S)-N-(1-{5-(Hydr oxym eth yl)-5-[(m eth oxy-
p h en oxy)m et h yl]oxola n -2-yl}-2-oxoh yd r op yr im id in -4-
yl)ben za m id e (r a c-5) a n d (2S,5R or 2R,5S)-N-(1-{5-(Hy-
d r oxym eth yl)-5-[(m eth oxyp h en oxy)m eth yl]oxola n -2-yl}-
2-oxoh yd r op yr im id in -4-yl)ben za m id e (r a c-6). The insepa-
rable mixture of diastereoisomers 4 (4.0 g, 7.38 mmol) was
dissolved in anhydrous CH2Cl2 (40 mL), cooled to -78 °C, and
treated with a solution of BCl3 in CH2Cl2 (1 M, 25 mL). After
stirring for 3 h, the reaction was quenched with MeOH (5 mL)
and warmed to room temperature. All of the volatiles were
removed under vacuum, and the residue was coevaporated
three times with MeOH (10 mL). The crude product was
purified by flash chromatography on silica gel using EtOAc/
hexanes (2:1 f pure EtOAc) to give diastereoisomer r a c-5
(0.75 g, 25% yield from 3) and diastereoisomer r a c-6 (1.43 g,
49% yield from 3) as foams.
Diastereoisomer r a c-5: 1H NMR (CDCl3) δ 8.55 (d, J ) 7.6
Hz, 1 H, H-6), 7.50-8.10 (m, 6 H, H-5, PhH), 6.95 (br s, 4 H,
ArH), 6.35 (m, 1 H, H-2′), 4.37 (d, J ) 10.1 Hz, 1 H, HOCH2
or ArOCH2), 4.11 (d, J ) 10.1 Hz, 1 H, HOCH2 or ArOCH2),
3.90 (s, 3 H, OCH3), 3.75 (s, 2 H, HOCH2 or ArOCH2), 2.85
(m, 1 H, H-3′a or H-4′a), 2.30 (m, 2 H, H-3′a,b or H-4′a,b), 2.10
(m, 1 H, H-3′b or H-4′b); FAB-MS (relative intensity) 452 (MH+,
17), 451 (M+•, 15), 216 (bH2+, 100).
Diastereoisomer r a c-6: 1H NMR (CDCl3) δ 8.38 (d, J ) 7.4
Hz, 1 H, H-6), 7.40-7.92 (m, 6 H, H-5, PhH), 6.80 (br s, 4 H,
ArH), 6.35 (dd, J ) 6.6, 4.3 Hz, 1 H, H-2′), 3.99 (d, J ) 11.7
Hz, 1 H, HOCH2 or ArOCH2), 3.91 (s, 2 H, HOCH2 or ArOCH2),
3.86 (d, J ) 11.7 Hz, 1 H, HOCH2 or ArOCH2), 3.80 (s, 3 H,
OCH3), 2.80 (m, 1 H, H-3′a or H-4′a), 2.21 (m, 2 H, H-3′a,b or
H-4′a,b), 2.15 (m, 1 H, H-3′b or H-4′b); FAB-MS (relative
intensity) 452 (MH+, 17), 451 (M+•, 15), 216 (bH2+, 100).
the â-D-dideoxyribo isomer separated by the lipase-
catalyzed resolution (data not shown). The results
presented here suggest, on the basis of the strong anti-
HIV activity of the 5′-triphosphate, en t-1h -TP , that the
2′,3′-dideoxy-4′-C-ethynyl-â-D-ribopentofuranosyl nucleo-
side template is an attractive scaffold for the develop-
ment of prodrugs capable of bypassing the first kinase.
These types of compounds may also offer an additional
advantage of having reduced toxicity because the lack
of 3′-OH prevents them from being good substrates for
cellular polymerases.
Gen er a l Exp er im en ta l Section
All chemical reagents were commercially available. Column
chromatography was performed on silica gel 60, 230-240 mesh
(E. Merck), and analytical TLC was performed on Analtech
1
Uniplates silica gel GF. Routine IR and H NMR spectra were
recorded using standard methods. Positive-ion fast-bombard-
ment mass spectra (FABMS) were obtained on a VG 7070E
mass spectrometer at an accelerating voltage of 6 kV and a
resolution of 2000. Glycerol was used as the sample matrix,
and ionization was effected by a beam of xenon atoms.
Elemental analyses were performed by Atlantic Microlab, Inc.,
Norcross, GA.
5-[(4-Met h oxyp h en oxy)m et h yl]-5-(p h en ylm et h oxy)-
m eth yl]oxola n -2-ol (2). This compound was reported in ref
10 but was not full characterized. We followed the same
procedure and obtained 2 as an oil (mixture of anomers): 1H
NMR (CDCl3) δ 7.30 (m, 5 H, PhH), 6.80 (m, 4 H, ArH), 5.51
(irregular t, 0.5 H, H-2), 5.46 (dd, J ) 7.5, 4.2 Hz, 0.5 H, H-2),
4.60 (AB q, J ) 11.8 Hz, 1 H, PhCH2O), 4.52 (AB q, J ) 12.3
Hz, 1 H, PhCH2O), 3.80 (AB q, J ) 9.2 Hz, 1 H, BnOCH2 or
ArOCH2), 3.74 (s, 3 H, OCH3), 3.64 (AB d, J ) 1.0 Hz, 1 H,
BnOCH2 or ArOCH2), 3.58 (d, J ) 7.5 Hz, 0.5 H, BnOCH2 or
ArOCH2), 3.46 (AB q, J ) 9.5 Hz, 1 H, BnOCH2 or ArOCH2),
3.12 (d, J ) 7.5 Hz, 0.5 H, BnOCH2 or ArOCH2), 1.90-2.20
(m, 4 H, H-3a,b and H-4a,b); FAB-MS (relative intensity) 344
(M+•, 89). Anal. Calcd for C20H24O5: C, 69.75; H, 7.02. Found:
C, 69.60; H, 7.06.
(2R,5R or 2S,5S)-6-Am in o-3-{5-eth yn yl-5-[(4-m eth oxy-
p h en oxy)m et h yl]oxola n -2-yl}-3-h yd r op yr im id in -2-on e
(r a c-9). A solution of r a c-5 (0.74 g, 1.63 mmol) and 1,3-
dicyclohexylcarbodiimide (DCC, 1.02 g, 4.93 mmol) in anhy-
drous DMSO (40 mL) was cooled to 0 °C and treated with
dichloroacetic acid (0.08 mL, 0.58 mmol). The resulting solu-
tion was stirred for 1.5 h while allowing it to reach room
temperature. The precipitate formed (dicyclohexylurea, DCU)
was filtered off and washed with EtOAc (50 mL), and the
combined filtrate was diluted with EtOAc (200 mL) and
extracted with water (3 × 100 mL) and brine (2 × 100 mL).
The organic layer was separated, dried (MgSO4), and concen-
trated under vacuum. The residue was purified by flash
chromatography on silica gel using EtOAc followed by CH2-
Cl2/MeOH (40:1 f 20:1) to give 0.68 g (93.1%) of aldehyde r a c-
7. Thin-layer chromatography on silica gel using CHCl3/n-
BuOH/acetone (40:25:7.5) revealed some contamination with
DCU, and the product was used immediately in the following
reaction. A suspension of (chloromethyl)triphenylphosphonium
chloride (2.15 g, 6.19 mmol) in anhydrous THF (50 mL) was
cooled to -78 °C and treated with a solution of n-BuLi in
hexanes (1.6 M, 4.0 mL). After 1 h of stirring, a solution of
aldehyde r a c-7 (0.68 g, 1.51 mmol) in dry THF (50 mL) was
added. The temperature of the bath was raised to 0 °C, and
stirring continued for 3 h. Following the cautious addition of
an aqueous saturated solution of H4NCl (10 mL), the reaction
mixture was extracted with EtOAc (2 × 100 mL). The
combined organic layer was washed with brine (2 × 75 mL),
dried (MgSO4), and concentrated under vacuum. The crude
product (r a c-8) was still contaminated with DCU and tri-
phenylphosphine oxide. Hence, it was used as such for the
following reaction. Crude r a c-8 was dissolved in anhydrous
THF (40 mL), cooled to -78 °C, and treated slowly with a
solution of n-BuLi in hexanes (1.6 M, 20 mL). After stirring
5-[(4-Met h oxyp h en oxy)m et h yl]-5-[(p h en ylm et h oxy)-
m eth yl]oxola n -2-yl a ceta te (3). A solution of lactol 2 (6.10
g, 17.7 mmol) in dry pyridine (90 mL) was treated with acetic
anhydride (4.2 mL, 44.4 mmol). After stirring at room tem-
perature for 72 h, the reaction mixture was diluted with EtOAc
(200 mL), washed with water (3 × 100 mL), dried (MgSO4),
and concentrated under vacuum. The crude residue was
purified by flash column chromatography on silica gel using
EtOAc/hexanes (1:6 f 1:3) to give 5.93 g (86.7%) of 3 as a clear
oil (mixture of anomers); 1H NMR (CDCl3) δ 7.25 (m, 5 H,
PhH), 6.80 (m, 4 H, ArH), 6.32 (m, 1 H, H-2), 4.50-4.60
(overlapping s and AB q, J ) 12.4 Hz, 2 H, PhCH2O), 3.80-
4.00 (2 AB q, J ) 9.4 Hz, 2 H, BnOCH2 or ArOCH2), 3.73 and
3.74 (s, 3 H, OCH3), 3.48-3.62 (overlapping s and AB q, J )
9.8 Hz, 2 H, BnOCH2 or ArOCH2), 1.90-2.30 (m, 4 H, H-3a,b
and H-4a,b), 1.93 and 1.94 (s, 3 H, OCOCH3); FAB-MS (relative
intensity) 387 (MH+, 25), 386 (M+•, 100). Anal. Calcd for
C
22H26O6: C, 68.37; H, 6.78. Found: C, 68.38; H, 6.77.
N -(1-{5-[(4-Me t h o x y p h e n o x y )m e t h y l]-5-[(p h e n y l-
m et h oxy)m et h yl]oxola n -2-yl}-2-oxoh yd r op yr im id in -4-
yl)ben za m id e (4). N4-Benzoylcytosine (5.30 g, 24.6 mmol) was
suspended in dry CH3CN (20 mL) and was treated with bis-
(trimethylsilyl)trifluoroacetamide (BSTFA, 20 mL, 75.3 mmol)
under argon. After 1 h of stirring, the reaction mixture became
homogeneous. The volatiles were removed under vacuum to
give the silylated base as a thick oil. Separately, a solution of
acetate 3 (6.29 g, 16.3 mmol) in dry ClCH2CH2Cl (100 mL)
was added to the silylated base followed by the slow addition
of trimethylsilyl trifluoromethanesulfonate (TMSOTf, 4.8 mL,
26.4 mmol). The reaction mixture was stirred at room tem-
perature for 20 h and was diluted with CH2Cl2 (100 mL). After
cooling to 0 °C, the reaction was quenched by the addition of
a saturated solution of NaHCO3. The suspension that was

4′-C-Ethynyl-2′,3′-Dideoxynucleoside Analogue
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5047
for 2 h, the reaction was quenched by the cautious addition of
an aqueous saturated solution of H4NCl (20 mL). The organic
layer was washed with brine (2 × 75 mL), dried (MgSO4), and
concentrated under vacuum. The crude product was purified
by flash chromatography on silica gel using CH2Cl2/MeOH
(40:1 f 10:1) to give r a c-9 (0.271 g, 48.5% yield from r a c-5)
as a yellowish foam; 1H NMR (CDCl3) δ 8.04 (d, J ) 7.8 Hz, 1
H, H-4), 6.80 (m, 4 H, ArH), 6.18 (dd, J ) 6.6, 3.1 Hz, H-2′),
6.08 (d, J ) 7.8 Hz, 1 H, H-5), 4.37 (d, J ) 10.9 Hz, 1 H,
ArOCH2), 4.10 (d, J ) 10.9 Hz, 1 H, ArOCH2), 3.80 (s, 3 H,
OCH3), 2.80 (m, 1 H, 1 H, H-3′a or H-4′a), 2.60 (s, 1 H, CCH),
10 days. The enzyme was removed by filtration through a pad
of Celite, and the solid cake was washed with the same solvent
mixture of CCl4 and CHCl3. The combined filtrate was
concentrated under reduced pressure, and the residue was
purified by flash column chromatography on silica gel (CHCl2/
MeOH, 40:1 f 20:1) to give 0.043 g (58%) of the less polar
monoacetate, en t-13 (91% ee, chiral HPLC, RT ) 22.38 min),
and a mixture of the more polar unreactive alcohols. The
mixture of unreactive alcohols was treated a second time under
the same conditions in CCl4/CHCl3 (1:1, 10 mL) with the
enzyme (0.10 g) and vinyl acetate (1 mL, 10.84 mmol). After
flash column chromatography on silica gel under the same
chromatographic conditions, 0.030 g of en t-12 (98% ee, chiral
HPLC, RT ) 14.32 min) was obtained.
2.46 (m, 1 H, 1 H, H-3′b or H-4′b), 1.98 (m, 2 H, H-3′a,b or
H-4′a,b); FAB-MS (relative intensity) 342 (MH+, 40), 112 (bH2
,
+
100). Anal. Calcd for C18H19N3O4‚0.25CH3OH: C, 62.73; H,
5.76; N, 12.03. Found: C, 63.05; H, 5.73; N, 11.63.
en t-13. (2S,5S)-N-{1-[5-Eth yn yl)-5-(a cetoxym eth yl)ox-
ola n -2-yl]-2-oxoh yd r op yr im id in -4-yl}ben za m id e. 1H NMR
(CDCl3) δ 8.10 (d, J ) 7.2 Hz, 1 H, H-6), 7.40-7.84 (m, 6 H,
H-5, Ph), 6.16 (dd, J ) 6.8, 2.6 Hz, 1 H, H-2′), 4.46 (d, J )
12.1 Hz, 1 H, CH2OH), 4.34 (d, J ) 12.1 Hz, 1 H, CH2OH),
2.84 (m, 1 H, H-3′a or H-3′b), 2.54 (s, 1 H, CCH), 2.00-2.20
(m, 3 H, H-3′a or H-3′b and H-4′a,b), 2.10 (s, 3 H, CH3CO).
en t-12: (2R,5R)-N-{1-[5-Eth yn yl)-5-(h yd r oxym eth yl)-
oxola n -2-yl]-2-oxoh yd r op yr im id in -4-yl}ben za m id e. The
1H NMR of this compound was identical to that of r a c-11.
Gen er a l P r oced u r e for th e Dea cyla tion of en t-12 a n d
en t-13. The compounds were suspended in a saturated metha-
nolic ammonia solution and kept at room temperature in a
sealed tube for 5 days. The solution was concentrated under
vacuum, and the solid obtained was purified first by flash
column chromatography on silica gel (CH2Cl2/MeOH, 10:1 f
6:1) to give a 70% yield of the final deblocked materials (en t-
1h and en t-14) as white foams. Both 1H NMR spectra were
identical to that of r a c-1h . en t-1h : [a]23D + 45.84 (c 0.59,
H2O). Anal. Calcd for C11H13N3O3‚0.33 H2O: C, 54.76; H, 5.70;
N, 17.41. Found: C, 54.52; H, 5.60; N, 17.33.
Ch ir a l HP LC Ch r om a togr a p h y. Chiralcel OD column
with hexanes/isopropyl alcohol (1:1) as the eluant; injection
volume 10 µL); flow rate 3.0 mL/min; UV detection at 220 nm.
Syn th eses of 5′-Tr ip h osp h a tes of r a c-1h , en t-1h , a n d
en t-14. The custom syntheses of these 5′-triphosphates were
performed by TriLink Biotechnologies, Inc. using conventional
methodology. r a c-1h -TP was 95.4% pure by HPLC and >95%
pure by 31P NMR; en t-1h -TP was 97.7% pure by HPLC and
>95% pure by 31P NMR; en t-14-TP was 97.08% pure by HPLC
and > 5% by 31P NMR. Additional information is provided as
Supporting Information.
n Oe Exp er im en ts. nOe buildups were not quantitative
because the samples were not degassed, but the qualitative
buildup data was adequate to determine the relative stereo-
chemistry. In r a c-10, the nOe data gave a distance between
H-5′a,b and H-1′ (nucleoside numbering) of 3.3 Å, whereas no
nOe was observed between the same set of protons in com-
pound r a c-1h . nOe buildup curves were obtained by irradiat-
ing the key target protons on both samples. An internuclear
distance of 2.4 Å for the distance from H-6 to H-5 (nucleoside
numbering) was used as a reference for the calculations. Data
points from the integrated nOe peaks were all normalized to
100% inversion of the target before the distance calculation
was carried out.
(2R,5R or 2S,5S)-6-Am in o-3-[5-et h yn yl-5-(h yd r oxy-
m eth yl)oxola n -2-yl]-3-h yd r op yr im id in -2-on e [(()-1-(2′,3′-
d id eoxy-4-C-eth yn yl-r ibo-p en tofu r a n osyl)cytosin e] (r a c-
1h ). A stirred solution of r a c-9 (0.261 g, 0.76 mmol) in
acetonitrile/water (4:1, 24 mL) was cooled to 0 °C and treated
with ammonium cerium (IV) nitrate (1.38 g, 2.51 mmol) for
2.5 h. The solvent was removed under vacuum, and the residue
was coevaporated with MeOH (3 × 10 mL). The crude product
obtained was purified once by flash chromatography on silica
gel with CH2Cl2/MeOH (10:1 f 3:1) and twice by reverse-phase
C-18 column chromatography eluting first with water and then
with MeOH/water (15:1 f 5:1). The combined fractions
containing the product were evaporated under vacuum and
then lyophilized to give 0.148 g (83%) of product r a c-1h .
Recrystallization from MeOH/ether provided a crystalline
1
solid, mp 166-167 °C; H NMR (D2O) δ 7.66 (d, J ) 7.61 Hz,
1 H, H-4), 6.80 (dd, J ) 7.5, 3.5 Hz, 1 H, H-2′), 5.90 (d, J ) 7.5
Hz, 1 H, H-5), 3.70 (AB q, J ) 12.3 Hz, 2 H, HOCH2), 2.88 (s,
1 H, CCH), 2.58 (m, 1 H, H-3′a or H-4′a), 2.05 (m, 3 H, H-3′b or
H-4′b plus H-3′a,b or H-4′a,b); FAB-MS (relative intensity)
236 (MH+, 35), 112 (bH2+, 100); HRMS (FAB) calcd for
C
11H14O3N3: 236.1035. Found: 236.1031.
(2S,5R or 2R,5S)-6-Am in o-3-[5-et h yn yl-5-(h yd r oxy-
m eth yl)oxola n -2-yl]-3-h yd r op yr im id in -2-on e [(()-1-(2′,3′-
d id eoxy-4-C-eth yn yl-r ibo-p en tofu r a n osyl)cytosin e] (r a c-
10). This compound was obtained following the same procedure
as described for the other diastereoisomer r a c-1h : 1H NMR
(D2O) δ 7.86 (d, J ) 8.0 Hz, 1 H, H-4), 6.04 (m, 1 H, H-2′),
5.96 (d, J ) 7.4 Hz, 1 H, H-5), 3.57 (AB q, J ) 11.7 Hz, 2 H,
HOCH2), 3.00 (s, 1 H, CCH), 2.45 (m, 1 H, H-3′a or H-4′a), 2.14
(m, 3 H, H-3′b or H-4′b plus H-3′a,b or H-4′a,b); FAB-MS (relative
intensity) 236 (MH+, 73), 112 (bH2+, 100); HRMS (FAB) calcd
for C11H14O3N3: 236.1035. Found: 236.1034.
(2R,5R or 2S,5S)-N-{1-[5-Eth yn yl)-5-(h yd r oxym eth yl)-
oxola n -2-yl]-2-oxoh yd r op yr im id in -4-yl}ben za m id e (r a c-
11). A solution of r a c-1h (0.085 g, 0.361 mmol) in anhydrous
pyridine (15 mL) was treated with chlorotrimethylsilane
(TMSCl, 0.35 mL, 2.57 mmol) and stirred at room temperature
for 20 min. Benzoyl chloride (0.23 mL, 1.93 mmol) was added,
and after 2 h, the reaction was cooled to 0 °C and quenched
with water (2 mL). After stirring for 15 min, concentrated NH4-
OH (5 mL) was added, the ice bath was removed, and the
solution was left at room temperature for 1 h. All volatiles were
removed under vacuum, and the residue was coevaporated
three times with MeOH (10 mL). The final residue was purified
first by flash column chromatography on silica gel (EtOAc/
hexanes, 2:1 f EtOAc) followed by a second purification
through a C-18 reversed-phase column (water and water/
MeOH, 6:1 f 1:4) to give 0.084 g (68.5%) of r a c-11 as a foam;
1H NMR (CDCl3) δ 8.25 (d, J ) 7.4 Hz, 1 H, H-6), 7.82 (d, J )
7.6 Hz, 1 H, H-5), 7.40-7.60 (m, 5 H, PhH), 6.14 (dd, J ) 7.0,
3.2 Hz, 1 H, H-2′), 4.01 (d, J ) 11.8 Hz, 1 H, CH2OH), 3.80 (d,
J ) 11.8 Hz, 1 H, CH2OH), 2.78 (m, 1 H, H-3′a or H-4′a), 2.50
(s, 1 H, CCH), 2.30 (m, 1 H, H-3′b or H-4′b), 2.20 (m, 1 H, H-4′a
or H-3′a), 2.80 (m, 1 H, H-4′b or H-3′b).
Sin gle-Cr yst a l X-r a y Diffr a ct ion An a lysis of r a c-1h .
C
11H13N3O3‚H2O, fw ) 253.26, triclinic P-1, a ) 5.535(1) Å, b
) 7.358(1) Å, c ) 15.176(4) Å. V ) 593.6(1) Å3, Z ) 2, Fcalcd
)
1.414 Mg/m3, λ(Cu KR) ) 1.54178 Å, µ ) 0.921 mm-1, F (000)
) 268, T ) 293(2) K. A colorless 0.39 × 0.28 × 0.06-mm3
crystal was used for data collection. The theta range for data
collection was 2.97 to 66.59° with 3510 reflections collected.
Additional information and tables of coordinates, bond dis-
tances and bond angles, and anisotropic thermal parameters
have been deposited with the Crystallographic Data Centre,
Cambridge, CB2, and 1EW, England.
Lip a se-Ca ta lyzed Resolu tion . A solution of r a c-11 (0.065
g, 0.191 mmol) in CCl4/CHCl3 (1:1, 20 mL) was treated with
vinyl acetate (1 mL, 10.84 mmol) and lipase PS-C Amano I
(0.190 g). The mixture was stirred at room temperature for
An tivir a l Activity in MT-2 a n d MT-4 Cells. MT-4 cells
(3 × 104) were exposed to 100 TCID50 of HIV-1LAI and were
cultured in the presence of r a c-1h and AZT and used as a
control. The EC50 values were determined using an MTT assay

5048 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Siddiqui et al.
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of 4′-cyanothymidine and analogues as potent inhibitors of HIV.
Tetrahedron Lett. 1992, 33, 37-40.
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Nucleosides and nucleotides. 183. Synthesis of 4 ′alpha- branched
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Lett. 1999, 9, 385-388.
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S.; Matsuda, A. Nucleosides and nucleotides. 185. Synthesis and
biological activity of 4′-alpha-C-branched-chain sugar pyrimidine
nucleosides. J . Med. Chem. 1999, 42, 2901-2908.
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on day 5 of the cultures. All assays were conducted in
duplicate. MT-2 cells (2 × 103) were exposed to 100 TCID50 of
HIV-1LAI and were cultured in the presence of r a c-1h and AZT.
The EC50 values were determined using an MTT assay on day
7 of the cultures. All assays were conducted in duplicate.
In h ibition of Wild -Typ e HIV-1 RT. The assay was done
in duplicate as previously described.16 Briefly, for each sample,
0.25 µg of single-stranded M13mp18 DNA (New England
Biolabs) was hybridized to 0.5 µL of 1.0 OD260/mL -47
sequencing primer (New England Biolabs). The template
primer was suspended in a solution containing 100.0 µL of 25
mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl2, 100.0 µg/mL
bovine serum albumin (BSA), 10.0 mM 3-[(3-cholamidopropyl)-
dimethylammonio-]-1-propanesulfonate (CHAPS), 2.0 mM
dithiothreitol, 10.0 µM each of dATP, dGTP, and dTTP, 5.0
µM dCTP, 2.0 µM [R-32P] dCTP, and the indicated concentra-
tion of inhibitor. Extension was initiated by the addition of
1.0 µg of wild-type HIV-1 RT or RT-variant M184V. The
mixture was incubated for 30 min at 37°, and the reaction was
halted by the addition of 3 mL of 10% trichloroacetic acid
(TCA). Precipitated DNA was collected by suction filtration
through Whatman GF/C glass filters. The amount of incorpo-
rated radioactivity was determined by liquid scintillation
counting. The amount of incorporated radioactivity in the
absence of inhibitor was considered 100% activity. The amount
of radioactivity incorporated in the presence of inhibitor was
normalized to this value.
(11) Ti, G. S.; Gaffney, B. L.; J ones, R. A. Transient protection:
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Am. Chem. Soc. 1982, 104, 1316-1319.
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M.; Blumberg, P. M.; Lee, J .; Marquez, V. E. Conformationally
constrained analogues of diacylglycerol. 24. Asymmetric syn-
thesis of a chiral (R)-DAG-lactone template as
a versatile
precursor for highly functionalized DAG-lactones. Org. Lett.
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reverse transcriptase involves steric hindrance with beta-
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(16) Boyer, P. L.; Gao, H. Q.; Clark, P. K.; Sarafianos, S. G.; Arnold,
E.; Hughes, S. H. YADD mutants of human immunodeficiency
Ack n ow led gm en t. We thank Dr. J ames A. Kelley
of the Laboratory of Medicinal Chemistry, NCI-Fred-
erick, CCR, for mass spectral data. The help of Mr.
David A. Luca, Mr. J ohn R. Klose, and Dr. Gwendolyn
N. Chmurny of the NMR Group, Laboratory of Pro-
teomics and Analytical Technologies, SAIC Frederick,
for 1D and 2D NMR experiments, structural minimiza-
tions, and peak assignments is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: For r a c-1h , crystal-
lographic data, atomic coordinates, bond lengths and angles,
ansiotropic displacement parameters, hydrogen coordinates
and isotropic displacement parameters, torsion angles, hydro-
gen bonds, and a PDB file. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Novel mutation in the human-immunodeficiency-virus type-1
reverse-transcriptase gene that encodes cross-resistance to 2′,3′-
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