Synthesis and evaluation of 2′-substituted cyclobutyl nucleosides and nucleotides as potential anti-HIV agents

Article abstract of DOI:10.1016/j.bmcl.2007.03.094

A series of 2′-substituted cyclobutyl nucleoside analogs were efficiently prepared by constructing the core cyclobutyl ring using different [2+2] cycloaddition approaches. The triphosphate derivative of a cyclobutyl nucleoside was also synthesized and eva

Full text of DOI:10.1016/j.bmcl.2007.03.094

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3398–3401
Synthesis and evaluation of 20-substituted cyclobutyl
nucleosides and nucleotides as potential anti-HIV agents
Yongfeng Li,a Shuli Mao,a Michael W. Hager,a Kimberlynne D. Becnel,a
Raymond F. Schinazib and Dennis C. Liottaa,*
aDepartment of Chemistry, Emory University, Atlanta, GA 30322, USA
bDepartment of Pediatrics, Laboratory of Biochemical Pharmacology, Veterans Affairs Medical Center,
Emory University School of Medicine, Decatur, GA 30033, USA
Received 10 February 2007; revised 19 March 2007; accepted 29 March 2007
Available online 3 April 2007
Abstract—A series of 20-substituted cyclobutyl nucleoside analogs were efficiently prepared by constructing the core cyclobutyl ring
using different [2+2] cycloaddition approaches. The triphosphate derivative of a cyclobutyl nucleoside was also synthesized and eval-
uated against wild-type and mutant HIV reverse transcriptases (RT). Whereas the nucleoside analogs were inactive against HIV-1 in
culture, the nucleotide showed good activity not only against wild-type and recombinant HIV RT (IC50 = 4.7, 6.9 lM), but also
against the M184I and M184V mutants (IC50 = 6.1, 6.9 lM) in cell-free assays.
Published by Elsevier Ltd.
Acquired immune deficiency syndrome (AIDS) has rap-
idly become one of the major causes of death in the
world. It is estimated that over 40 million people have
developed human immunodeficiency virus (HIV) infec-
tions, which is the causative agent of AIDS.1 In 1985,
30-azido-30-deoxythymidine (AZT) was approved as
the first synthetic nucleoside to inhibit the replication
of HIV. Since then, a number of other synthetic nucleo-
side analogs have been proven to be effective against
HIV. After cellular phosphorylation to the triphosphate
form by cellular kinases, the nucleotides are incorpo-
rated into a growing strand of viral DNA and cause
chain termination due to the absence of the 30-hydroxyl
group.
steric hindrance, which would prevent further incorpo-
ration of nucleoside analogs such as 3TC and FTC in
the nucleotide forms.3
Despite the enormous success of nucleoside based ther-
apy of HIV infection, there is still no cure for AIDS.
One reason is that the prolonged clinical use of nucleo-
side analogs gives rise to resistant viruses that contain
mutations in the RT.2 For example, the M184V/I muta-
tion in HIV-1 RT is associated with high level resistance
to lamivudine (3TC) and emtricitabine (FTC). Struc-
tural studies suggest that the mechanism of resistance
of HIV-1 RT carrying the M184V/I mutation involves
We postulated that the more rigid and smaller cyclobu-
tyl ring would enable the nucleoside analog to fit into
the more sterically hindered active site of RT containing
the M184V/I mutants. Nucleoside analogs of this type
received a great deal of attention several years ago with
the discovery of naturally occurring Oxetanocin A,
which shows activity against HIV and Lobucavir
(Cyclobut-G) with activity against HBV. However,
virtually all of the reported derivatives possess the
Keywords: Antiviral; Anti-HIV; Nucleoside; Analogs; Resistance;
Synthesis.
*
Corresponding author. E-mail: dliotta@emory.edu
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.03.094

Y. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3398–3401
3399
2030-bis-hydroxyl motif that enables them to be phos-
phorylated and incorporated into growing strands of
DNA. We were interested in preparing a series of 20-
substituted cyclobutyl nucleosides that only have the
30-hydroxymethyl for phosphorylation, but not the 20-
hydroxymethyl motif. We reasoned that these nucleo-
side analogs could fit into the more sterically hindered
active site of M184I/V mutant forms of RT.
with L-Selectride in THF solution gave cis-compound
5, which was converted to trans-(3-benzyloxymeth-
yl)cyclobutanol 6 via Mitsunobu reaction followed by
hydrolysis of the resulting ester. Cyclobutanol 6 reacted
with N3-benzoyl-5-fluorouracil under Mitsunobu condi-
tions to give nucleoside 7 which was deprotected to give
5-fluorouracil nucleoside 8 in moderate yield. Finally,
compound 8 was converted to the corresponding 5-flu-
orocytosine derivative 1 by the procedure shown in
Scheme 2.
In this paper, we report the synthesis and biological
evaluation of 20-substituted cyclobutyl nucleosides as
potential anti-HIV agents. To elucidate the mechanism
of action of these novel cyclobutyl nucleosides, a tri-
phosphate derivative 21 was also synthesized and evalu-
ated against wild-type and mutant HIV-1 RT.
Fluorine as a substituent is isosteric with hydrogen;
therefore, it should not affect the spatial disposition of
atoms in the molecule. On the other hand, the phys-
ico-chemical properties of a fluorine atom can, due to
its high electron negativity, profoundly affect the electro-
static properties of the bonds around it, thereby influ-
encing both the conformation of the four-membered
ring and the innate strength of the bonds.
The 5-fluoro-1-[cis-3-(hydroxymethyl)-cyclobutyl]-cyto-
sine 1 was synthesized using a reported procedure.4 As
shown in Scheme 1, reduction of the cyclobutanone 4
Scheme 1. Synthesis of 5-fluoro-1-[cis-3-(hydroxymethyl)-cyclobutyl]-uracil.
Scheme 2. Synthesis of 20-substituted cyclobutyl 5-fluorocytidine analogs.

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Y. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3398–3401
Scheme 3. Synthesis of 3-benzyloxymethyl-2-methylcyclobutanol.
In the synthesis of 20-fluoro-substituted cyclobutyl
nucleosides, we found electrophilic fluorination with
Selectfluor to be the most efficient method to introduce
fluorine in the cyclobutanone 4.5 As shown in Scheme 2,
the cyclobutanone 4 was first deprotonated by LDA,
followed by lithium enolate quenching with TMSCl to
afford the silyl enol ether 9.6 This silyl enol ether was
then allowed to react with Selectfluor to give a 3:1 mix-
ture of inseparable diastereomers 10. The crude mixture
was reduced by L-Selectride followed by chromatogra-
phy to isolate pure diastereomer 11 in good yield. Mesy-
lation of 11 gave compound 12, which then was coupled
with N3-(4-methoxybenzyl)-5-fluorouracil under basic
conditions to give 53% of nucleoside 13. Removal of
both the p-methoxylbenzyl and benzyl groups by alumi-
num trichloride afforded compound 14 in 80% yield. The
stereochemistry of 14 was confirmed by X-ray crystal
structure analysis.7 Conversion of 14 to 5-fluoro-1-
[trans-2-fluoro-cis-3-(hydroxylmethyl)-cyclobutyl]cyto-
sine 2 was achieved in the standard three-step process as
shown in Scheme 2.
Scheme 4. Synthesis of triphosphate of compound 1.
Table 1. Comparison of inhibition of HIV RT in cell-free assays
HIV RTa
Inhibition of RT activity
(IC50, lM)
3TC-TP
Compound 21
Recombinant HIV RT (WT)
HIV RT (WT)
HIV RT (M184I)
3.0
6.5
4.7
6.9
6.1
6.9
To compare the electronic properties of the 20-substi-
tuted analogs, the 20-methylcyclobutyl nucleoside was
synthesized and evaluated. As shown in Scheme 3, a ket-
eniminium salt was used as the key intermediate to form
the cyclobutanone.8 The keteniminium triflate generated
from N,N-dimethylpropionamide in situ reacted with al-
lyl benzyl ether to give the intermediate cyclobutanimin-
ium salt which is hydrolyzed to provide cyclobutanone
15 as the major diastereomer with the methyl group
trans to the benzyl group (dr = 9:1). Reduction of the
cyclobutanone 15 gave primarily one diastereomer 16
with the methyl group and the hydroxyl group cis to
each other as confirmed by 1H NOE analysis. The
remainder of the synthesis of 5-fluoro-1-[trans-20-
methyl-cis-30-(hydroxymethyl)cyclobutyl]cytosine 3 is
shown in Scheme 2.
>10
>10
HIV RT (M184V)
a All the HIV RT used, except the recombinant RT, was obtained from
viral lysates from PBM cell infected with respective HIV.
100 lM, its triphosphate 21 exhibited comparable anti-
HIV activity to 3TC-TP against recombinant HIV RT
and wild-type HIV RT (IC50 = 4.7, 6.9 lM). Further-
more, the triphosphate showed also very good activity
against M184I and M184V mutant RT (IC50 = 6.1,
6.9 lM), which were not inhibited by 3TC-TP. The re-
sults suggest that the cyclobutyl nucleotide can be incor-
porated into the DNA chains of HIV RT and M184M/V
mutants to terminate the DNA elongation.
Although they were not found to be toxic, unfortu-
nately, none of the nucleosides 1, 2, and 3 showed signif-
icant anti-HIV activity up to 100 lM in primary human
lymphocytes infected with HIV-1 (strain LAI). To eluci-
date the mechanism of the nucleotides incorporating
into the viral DNA chain, the triphosphate of nucleoside
1 was synthesized according to Scheme 4.
All the biochemical results were obtained according to
the assay described by Eriksson et al.10 and are shown
in Table 1.
In conclusion, several 20-substituted cyclobutyl nucleo-
sides were synthesized and evaluated as anti-HIV agents.
Although the cyclobutyl nucleosides were not active
against HIV, the triphosphate form of one of them was
quite active against wild-type and M184V or M184I
HIV RT. This suggests that the nucleosides are not being
phosphorylated by the cellular kinases. It appears that
the cellular kinases cannot recognize the modified nucle-
oside analogs due to their structural and conformational
differences from the natural nucleoside substrates. Our
future work will be focused on the synthesis of phosphate
prodrugs of various cyclobutyl nucleoside analogs to cir-
cumvent the problems with phosphorylation.
The monophosphate 20 was generated by the reaction of
the nucleoside with phosphorus oxychloride using trim-
ethylphosphate as the solvent. The monophosphate 20
was activated as its morphine phosphoramidate, which
was subsequently combined with nucleophilic pyrophos-
phate to provide triphosphate 21 (Scheme 4).9–11
Although the previously described anti-HIV studies
demonstrated that compound 1 was not active up to

Y. Li et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3398–3401
3401
31; (c) Stavber, S.; Jereb, M.; Zupan, M. Synthesis 2002,
17, 2609; (d) Stavber, G.; Zupan, M.; Jereb, M.; Stavber,
S. Org. Lett. 2004, 6, 4973.
Acknowledgments
This work was supported by NIH Grants 2R37AI-28731
(D.C.L.), 1R37AI-41890 (R.F.S.), Emory’s CFAR
Grant 5P30-AI150409, and the Department of Veterans
Affairs (R.F.S.). We thank Dr. R. Pai and Kim L. Rapp
for performing the RT assays.
6. Vidal, J.; Huet, F. J. Org. Chem. 1988, 53, 611.
7. X-ray structure of 5-fluoro-1-[trans-2-fluoro-cis-3-
(hydroxymethyl)cyclobutyl]uracil 14 (CCDC 640733):
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´
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