Synthesis of nucleoside 5′-O-α,β-methylene-β- triphosphates and evaluation of their potency towards inhibition of HIV-1 reverse transcriptase

Article abstract of DOI:10.1039/b922846b

A polymer-bound α,β-methylene-β-triphosphitylating reagent was synthesized and subjected to reactions with unprotected nucleosides, followed by oxidation, deprotection of cyanoethoxy groups, and acidic cleavage to afford nucleoside 5′-O-α,β-methylene-β-tr

Full text of DOI:10.1039/b922846b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of nucleoside 5¢-O-a,b-methylene-b-triphosphates and evaluation of
their potency towards inhibition of HIV-1 reverse transcriptase†
Y. Ahmadibeni,a,c C. Dash,b M. J. Hanley,a S. F. J. Le Grice,d H. K. Agarwala and K. Parang*a
Received 2nd November 2009, Accepted 21st December 2009
First published as an Advance Article on the web 13th January 2010
DOI: 10.1039/b922846b
A
polymer-bound
a,b-methylene-b-triphosphitylating
create the primer for plus strand DNA synthesis.20 Both DNA
polymerase and RNase H activities of HIV-1 RT have been
considered as potential targets for antiretroviral therapy. 3¢-
Substituted modified nucleoside triphosphate analogues (e.g., 3¢-
azidodeoxythymidine triphosphate) are incorporated into DNA,
behave as chain terminators, and inhibit DNA synthesis by HIV-
1 RT. On the other hand, the discovery of potent and selective
inhibitors of HIV-1 RNase H has been challenging, and inhibitors
of this enzyme function have yet to reach clinical development
stage.18 Although there are more than a dozen FDA approved
drugs that inhibit the polymerase function of HIV-1 RT, there is
not a single RNase H inhibitor in clinics. Therefore, a potential
RNase H inhibitor can serve as a mechanistic probe and/or a lead
compound against HIV-1 RT. Nucleotide analogues have been
described as inhibitors of HIV-1 RNase H activity.21,22
Less attention has been given to the synthesis of modified
nucleotides containing branched triphosphate groups that can
have diverse applications in studying the enzymes (e.g., RNase H)
that use natural nucleoside triphosphates. Previously, we reported
the synthesis of 5¢-O-nucleoside b-triphosphates using a solid-
phase b-triphosphitylating reagent.23 Herein, we report the solid-
phase synthesis of 5¢-O-nucleoside b-triphosphates containing
a,b-methylene triphosphate bridge by using a novel solid-phase
phosphitylating reagent. The unusual branched triphosphate in
nucleoside triphosphate analogues may be useful in elucidation of
enzymatic functions and mechanism. Furthermore, the presence
of an a,b-methylene group in the modified nucleotide may provide
more stability to cleavage by cellular hydrolytic enzymes. We
determined the potency of the modified analogues towards HIV-1
RT polymerase and RNase H activity.
reagent was synthesized and subjected to reactions with
unprotected nucleosides, followed by oxidation, deprotection
of cyanoethoxy groups, and acidic cleavage to afford
nucleoside 5¢-O-a,b-methylene-b-triphosphates. Among all
the compounds, cytidine 5¢-O-a,b-methylene-b-triphosphate
inhibited RNase H activity of HIV-1 reverse transcriptase
with a Ki value of 225 lM.
Modified nucleoside triphosphates have received much attention
as mimics of naturally occurring deoxyribo- and ribonucleoside
triphosphates, as probes in several biochemical pathways involving
DNA and RNA synthesis, and as potential diagnostic and thera-
peutic agents.1,2 The structural similarity of modified nucleotides
to natural nucleoside triphosphates make them useful reagents
as substrates or inhibitors for DNA or RNA polymerases.3,4
Although most natural polymerase enzymes incorporate natural
nucleoside triphosphates into nucleic acids, there are certain poly-
merases that are capable of incorporating unnatural nucleoside
triphosphates into nucleic acids.5–7
A number of approaches have been focused on modifications
and/or substitution on the base,8–9 carbohydrate,10–13 and linear
triphosphate moieties14–17 to design modified nucleotides for
diverse applications in nucleic acid and antiviral research.
Early in the life cycle of human immunodeficiency virus type
1 (HIV-1), viral RNA is reverse transcribed into double stranded
DNA for integration into the genome of the infected cell.18 This
process is catalyzed by reverse transcriptase (RT), a virus encoded
heterodimeric enzyme composed of 66 and 51 kD subunits
(p66 and p51), possessing DNA polymerase and ribonuclease
H (RNase H) activities.19 DNA polymerase activity is required
for the synthesis of RNA : DNA heteroduplex from the single
stranded viral RNA. Whereas, RNase H activity is responsible
for hydrolyzing the RNA strand of RNA : DNA heteroduplex
molecules that are generated during reverse transcription and
a,b-Methylene-b-triphosphitylating reagent was first immo-
bilized on polystyrene resin-bound linker of p-acetoxybenzyl
alcohol. Coupling reactions of unprotected nucleosides with the
immobilized reagent followed by oxidation, deprotection, and
cleavage afforded nucleoside 5¢-O-a,b-methylene-b-triphosphates.
The advantages of this solid-phase strategy included major 5¢-
O-monophosphitylation, the facile isolation and purification of
products, removal of unreacted reagents and starting materials
in each step, and trapping of the linker on the resin in the final
cleavage reaction.
aDepartment of Biomedical and Pharmaceutical Sciences, College of Phar-
macy, The University of Rhode Island, Kingston, Rhode Island 02881, USA.
E-mail: kparang@uri.edu; Fax: +1-401-874-5787; Tel: +1-401-874-4471
bCentre for AIDS Health Disparities Research, Meharry Medical College,
Nashville, TN 37208, USA
Scheme
1 illustrates the synthesis of a,b-methylene b-
cDepartment of Chemistry, Columbus State University, Columbus, Georgia
31907, USA
triphosphitylating reagent 6. The reaction of bis(dichloro-
phosphino)methane (1) with diisopropylamine (1 equiv) in the
presence of 2,6-lutidine (1 equiv) in anhydrous THF afforded
2. Because of the bulkiness of the N,N-diisopropylamine group,
the replacement of –Cl groups in the less hindered phosphorous
group in 2 is significantly more desirable. Reaction of 2 with
3-hydroxypropionitrile (2 equiv) in the presence of 2,6-lutidine
dResistance Mechanism Laboratory, HIV Drug Resistance Program, Na-
tional Cancer Institute at Frederick, National Institute of Health, Frederick,
Maryland 21702, USA
† Electronic supplementary information (ESI) available: Experimental
procedures, characterization of resins with IR and final compounds with
NMR, high-resolution mass spectrometry, and quantitative phosphorus
analysis, DNA polymerase assay results. See DOI: 10.1039/b922846b
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Scheme 1 Synthesis of a,b-methylene-b-triphosphitylating reagent 6.
(2 equiv) afforded 3, which was subjected to reaction with water
(1 equiv) and 2,6-lutidine (1 equiv) to produce the intermediate 4.
presence of 5-(ethylthio)-1H-tetrazole gave 9a–f. Oxidation with
t-butyl hydroperoxide followed by removal of the cyanoethoxy
group with DBU, afforded the corresponding polymer-bound nu-
cleosides 5¢-O-a,b-methylene-b-triphosphosphate diester deriva-
tives, 11a–f. The cleavage of polymer-bound compounds was
carried out under acidic conditions, DCM/TFA/water/EDT
(72.5 : 23 : 2.5 : 2 v/v/v/v). The intramolecular cleavage mecha-
nism of final products from 12a–f is shown in Scheme 2. The
linker-trapped resin (13) was separated from the final products by
filtration and was converted to 14 by reaction with potassium
hydroxide. The crude products had a purity of 68–83% and
were purified on the C18 Sep-Pak cartridges to afford 5¢-O-
nucleoside a,b-methylene-b-triphosphates (15a–f, Scheme 2) in
47–73% overall yield (calculated from 8, 212–344 mg, Table S1,
see the Supporting Information†).
In
a separate reaction, phosphorus trichloride was re-
acted with 3-hydroxypropionitrile (1 equiv) in the presence
of 2,6-lutidine (1 equiv) in anhydrous THF to yield 2-
cyanoethoxyphosphorodichloridite (5), which was reacted with
4 (1 equiv) to yield a,b-methylene-b-triphosphitylating reagent
6, which was immediately used in the coupling reaction with
polymer-bound p-acetoxybenzyl alcohol 7. The chemical structure
of 6 was confirmed by high-resolution time-of-flight electrospray
mass spectrometry (ESI-TOF), but obtaining NMR for this
compound was not feasible because of its rapid decomposition.
Compound 6 was immediately used for the next coupling reaction
with polymer-bound p-acetoxybenzyl alcohol 7.
We
have
previously
reported
the
synthesis
of
aminomethylpolystyrene resin-bound p-acetoxybenzyl alcohol
7 (Scheme 2).24 Our earlier studies revealed the application of
polymer-bound p-acetoxybenzyl alcohol linkers in the synthesis
of a diverse number of compounds.25 Polymer-bound linker 7 was
selected for immobilization of reagent 6.
Scheme 2 shows the synthesis of nucleoside 5¢-O-a,b-methylene-
b-triphosphates. Aminomethyl polystyrene resin-bound linker
of p-acetoxybenzyl alcohol 7 was reacted with reagent 6 in
the presence of 2,6-lutidine to produce the corresponding
polymer-bound a,b-methylene-b-triphosphitylating reagent (8).
The treatment of excess of unprotected nucleosides (adenosine (a),
3¢-azido-3¢-deoxythymidine (b), 3¢-fluoro-3¢-deoxythymidine (c),
2¢,3¢-didehydrothymidine (d), cytidine (e), and inosine (f)) in the
Nucleoside 5¢-O-a,b-methylene-b-triphosphates were synthe-
sized with high selectivity as a result of this sequence because of
the presence of the phosphitylating reagent on the solid support
having a hindered structure, thereby allowing for the regioselective
reaction for unprotected nucleosides (e.g., adenosine, inosine, and
cytidine). The most reactive hydroxyl group of unprotected nucle-
osides reacted selectively with hindered polymer-bound reagents
when an excess of nucleoside was used. There was no need to
protect the free amino group in adenosine and cytidine. The
compounds were characterized by 1H NMR, 13C NMR, 31P NMR,
ESI-TOF, and phosphorus elemental analysis. 31P NMR indicated
the presence of only one diastereoisomer, suggesting that one of
the diastereoisomers of each compound was purified from the
Scheme 2 Synthesis of nucleoside 5¢-O-a,b-methylene-b-triphosphates 15a–f using polymer-bound linker 7.
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crude as the major product. On the other hand, there is a slight
possibility that diastereoisomers may not be distinguishable by 31
NMR because of overlapping peaks.
P
Purification of HIV-1 RT and enzyme assays was performed
according to the previously reported procedures.26–28 Initially, these
compounds were screened towards the polymerase and RNase H
activity of HIV-1 RT at a fixed concentration of the compound (1
mM). In comparison to the wild type enzyme, the polymerase
activity was not affected in the presence of these compounds
15a–f, however a few compounds exhibited inhibitory activity
against RNase H function. The results of the polymerase assay
are presented in Figure S1 (see Supporting Information†).
The results of the RNase H analysis are presented in Fig. 1A.
Lane W represents the RNase H cleavage products representative
of the primary and secondary cleavages in the absence of any
inhibitor. Lanes a–f represent the RNase H cleavage products
in the presence of the compounds 15a–f, respectively. Although
compounds 15a–d and 15f did not show any inhibitory activity,
the RNase H cleavage of HIV-1 RT was inhibited by 5¢-O-
cytidine a,b-methylene-b-triphosphate (15e) (Fig. 1A, Lane e).
To further evaluate the inhibitory potency of 15e, RNase H assay
was performed at increasing concentrations of 15e. Since RNase
H inhibition was not observed up to 100 mM concentration, the
results from 100 mM–1mM are presented in Fig. 1B.
Fig. 2 Inhibition kinetics analyses of compound 15e with RNase H
of HIV-1 RT. The enzyme activity was estimated using the substrate at
200 nM (ꢀ) and 400 nM (ꢀ) at increasing concentrations of 15e. Rate of
reaction represents rate of RNase H hydrolysis. Reciprocals of the rate of
hydrolysis were plotted versus the inhibitor concentrations. The straight
lines indicated the best fit of the data obtained. The inhibition constant Ki
was calculated from the point of the intersection of the plots.
To the best of our knowledge, this is the first report of the
synthesis of nucleoside 5¢-O-a,b-methylene-b-triphosphates. This
solid-phase methodology using novel polymer-bound phosphityl-
ating reagent (8) allowed for the expeditious development of
these analogues in a short synthetic route without the need for
the nucleoside phosphate precursors or protected nucleosides.
The solid-phase strategy offered the advantages of monosubsti-
tution, high selectivity, and facile isolation and purification of
products. Further exploring and optimization of 5¢-O-cytidine
a,b-methylene-b-triphosphate is required to design cell-permeable
compounds that can potentially inhibit the RNase H activity of
HIV-1 RT at lower concentrations.
Acknowledgements
We acknowledge the financial support from National Science
Foundation, Grant Number CHE 0748555. The work at NCI
was supported in part by the Intramural Research Program of the
NIH, NCI and Center for Cancer Research.
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