Solid-phase synthesis of 5′-O-β,γ-methylenetriphosphate derivatives of nucleosides and evaluation of their inhibitory activity against HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.tetlet.2010.04.005

Bis(dichlorophosphino)methane was converted to a β,γ-methylenetriphosphitylating reagent. The reagent was immobilized on aminomethyl polystyrene resin-bound linker of 4-acetoxy-3-phenylbenzyl alcohol to afford a polymer-bound β,γ-methylenetriphosphitylating reagent, which was reacted with unprotected nucleosides followed by oxidation with tert-butyl hydroperoxide, deprotection of cyanoethoxy groups with DBU, and acidic cleavage to produce 5′-O-β,γ-methylene triphosphate nucleosides in 53-82% overall yields. Among all the compounds, cytidine 5′-O-β,γ-methylenetriphosphate inhibited completely RNase H activity of HIV-1 reverse transcriptase at 700 μM.

Full text of DOI:10.1016/j.tetlet.2010.04.005

Tetrahedron Letters 51 (2010) 3010–3013
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
0
Solid-phase synthesis of 5 -O-b,
c-methylenetriphosphate derivatives
of nucleosides and evaluation of their inhibitory activity against HIV-1
reverse transcriptase
a,b
c
d
a,*
Yousef Ahmadibeni , Chandravanu Dash , Stuart F. J. Le Grice , Keykavous Parang
a
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, RI 02881, USA
Department of Chemistry, Columbus State University, Columbus, GA 31907, USA
Centre for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA
Resistance Mechanism Laboratory, HIV Drug Resistance Program, National Cancer Institute at Frederick, National Institute of Health, Frederick, MD 21702, USA
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Bis(dichlorophosphino)methane was converted to a b,c-methylenetriphosphitylating reagent. The
reagent was immobilized on aminomethyl polystyrene resin-bound linker of 4-acetoxy-3-phenylbenzyl
Received 9 March 2010
Revised 31 March 2010
Accepted 1 April 2010
Available online 9 April 2010
alcohol to afford a polymer-bound b, -methylenetriphosphitylating reagent, which was reacted with
c
unprotected nucleosides followed by oxidation with tert-butyl hydroperoxide, deprotection of cyanoeth-
0
oxy groups with DBU, and acidic cleavage to produce 5 -O-b,
8
c
-methylene triphosphate nucleosides in 53–
-methylenetriphosphate inhibited com-
0
2% overall yields. Among all the compounds, cytidine 5 -O-b,
c
pletely RNase H activity of HIV-1 reverse transcriptase at 700
l
M.
Ó 2010 Elsevier Ltd. All rights reserved.
Phosphate transfer is involved in several enzyme-catalyzed
tively. (ii) The coupling reactions are mostly carried out in anhy-
drous organic solvents. Because of the poor solubility of most
precursor phosphates in the reaction mixture, the yields are usu-
ally low. (iii) Stepwise lengthy purification of intermediates and fi-
nal products from the reagents are required. (iv) These strategies
require protection and deprotection reactions for most of the
1
–3
reactions
and therefore is a subject of considerable interest in
biological systems. Triphosphate mimics, such as methylenetri-
phosphates, halogenated methylenetriphosphates, and imidotri-
phosphates, have been used to probe the mechanism of
2–4
phosphoryl transfer in enzyme-catalyzed processes and to target
specific receptors or enzymes that bind or hydrolyze triphos-
nucleosides. Thus, alternative convenient strategies for the prepa-
5
–8
0
phates.
Replacement of labile P–O–P bond in nucleoside triphos-
–P bond in nucleotide analogs
ration of 5 -O-b,
c-methylenenucleoside triphosphates are highly
phates with a stable isosteric P–CH
2
desired.
results in enhanced metabolic stability. Synthesis of non-hydrolyz-
able triphosphate analogs of nucleosides is considered a challenge.
Herein, we report for the first time a solid-phase strategy for the
synthesis of b, -methylenetriphosphate nucleoside analogs by
using novel solid-supported phosphitylating reagent. b,
-methylenetriphosphitylating reagent was prepared first.
c
A number of solution-phase strategies have been previously
a
A
0
reported for the synthesis of nucleoside 5 -O-b,
c-methylenetri-
c
phosphates including some of the compounds described here by
the coupling reactions of nucleoside 5 -monophosphate salt forms
Aminomethyl polystyrene resin-bound linker of p-acetoxybenzyl
alcohol was used as solid support for immobilization of b,
0
14
or activated nucleoside monophosphates with diphosphonates
c-methylenetriphosphitylating reagent. The unprotected nucleo-
(
methylene diphosphonic acids). Some examples include the reac-
sides were reacted with the immobilized reagent. Subsequent
oxidation, deprotection, and cleavage afforded the b,c-methylene-
triphosphate nucleoside analogs.
0
tions of methylene diphosphonic acids with nucleoside 5 -mono-
phosphates,
5
Michael–Arbuzov reactions on ethyl(bishalomethyl) phosphinates
were used for the synthesis of the bismethylene triphosphates.
These synthetic strategies have been encountered by one or more
of the following difficulties. (i) Activated nucleoside monophos-
phates or their specific salt forms are required for the synthesis
of 5 -O-b,
9
,10
0
11,12
5 -monophosphate-N-methylimidazolides,
or
0
13
-monophosphate-imidazolides.
Alternatively,
sequential
Scheme 1 illustrates the synthesis of b,c-methylenetriphosph-
8
itylating reagent 6. Phosphorus trichloride (1, 6 mmol) was reacted
with diisopropylamine (2 equiv) to yield bis[diisopropyl-
amino]phosphorochloridite (2). The subsequent reaction of 2 with
water (1 equiv) afforded the intermediate bis[diisopropyl-
amino]hydroxyphosphine (3).
0
c-methylenetriphosphate nucleoside analogs, respec-
In
6 mmol) was treated with 3-hydroxypropionitrile (3 equiv) to
yield 5, which was reacted with 3 (1 equiv) to yield b, -meth-
ylenetriphosphitylating reagent (6). The chemical structure of 6
a separate reaction, bis(dichlorophosphino)methane 4
(
c
*
Corresponding author. Tel.: +1 401 874 4471; fax: +1 401 874 5787.
E-mail address: kparang@gmail.com (K. Parang).
0
040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.005

Y. Ahmadibeni et al. / Tetrahedron Letters 51 (2010) 3010–3013
3011
was confirmed by high-resolution time-of-flight electrospray mass
spectrometry (ESI-TOF) and elemental phosphorus analysis. Com-
pound 6 was immediately used for the next coupling reaction with
Cl
N
HO
N
iPr NH (2 eq)
2,6-lutidine, THF
P
N
H2O (1 eq)
P
N
2
PCl
3
1
2
,6-lutidine, THF
polymer-bound p-acetoxybenzyl alcohol
7
to avoid any
decomposition.
2
3
Aminomethyl polystyrene resin-bound linker of p-acetoxyb-
enzyl alcohol (7) was synthesized from aminomethyl polysty-
rene resin in multiple-step reactions and was selected as a
solid support for immobilization of the b, -methylenetriphosph-
itylating reagent. We have previously reported the application of
the p-acetoxybenzyl alcohol linkers for the solid-phase synthesis
NC
1
4
HO
CN
O
Cl
c
Cl
Cl
(3 eq)
3 (1 eq)
,6-lutidine, THF
P
P
P
P
2
Cl
Cl 2,6-lutidine, THF
O
4
O
of diverse number of compounds, such as carbohydrate and
CN CN
15–17
5
nucleoside monophosphates,
diphosphates, diphosphodithio-
1
8
NC
ates, triphosphates, or triphosphotrithioates, nucleoside b-tri-
O
O
1
9
P
P
P
N
N
phosphates,
oligodeoxynucleotides containing internucleotide
2
0
14
0
0
O
O
diphosphodiester linkages, sulfonamides, symmetrical 5 ,5 -
21
6
dinucleoside mono-, di-, tri-, and tetraphosphodiesters,
and
CN
CN
0
22
nucleoside 5 -O-a,b-methylene-b-triphosphates.
Scheme 2 shows the synthesis of nucleoside
a
,b- and b,
c-
Scheme 1. Synthesis of b,
c
-methylenetriphosphitylating reagent 6.
methylenetriphosphates. Polymer-bound linker 7 was reacted with
O
CN
OH
6
, 5-(ethylthio)-1H-
tetrazole
THF
O
HN
O
O
P
N
P
P
CN
O
7
AcO
P
8
CN
ROH
-(Ethylthio)-
H-tetrazole
CN
CN
5
CN
O
O
O
O
1
O
tBuOOH
THF
O
DBU
THF
P
P
O
O
P
P
P
CN
RO
O
O
THF/DMSO
b, c, e) or
DMSO (a, d)
O
RO
(
NC
NC
9a-e
10a-e
O
O
P
O
O
P
HN
O
O
O
O
OH
TFA/DCM
O
O
P
OH
P
P
P OH
OH H O/EDT
RO
2
O
O
HO
OR OH OH
H
1
1a-e
H
O
O
HN
HN
H O, KOH
2
OH
1
2
O
Dioxane
13
HO
O
O
P
O
P
RO
O
P
O
OH
OH OH OH
14a-e
NH2
N
N
O
N
N
N
NH
O
O
P
O
P
O
O
O
HO
P
O
O
O
HO
P
P
OH
O
P
O
O
OH OH OH
OH
OH
OHOH
N
3
1
4a
14b
O
NH2
N
N O
NH
O
O
P
O
P
O
P
O
P
O
P
O
N
HO
O
O
HO
O
P
O
O
O
OH OH OH
OH OH OH
OH
OHOH
14c
14d
OH
N
N
N
N
O
O
O
HO
P
P
OH
O
P
O
O
OH
OH
OHOH
14e
ROH: (a) adenosine, (b) 3'-azido-3'-deoxythymidine , (c) thymidine, (d) cytidine, (e) inosine
0
Scheme 2. Synthesis of nucleoside 5 -O-b,
c
-methylenetriphosphates 14a–e using polymer-bound reagent 8.

3
012
Y. Ahmadibeni et al. / Tetrahedron Letters 51 (2010) 3010–3013
Table 1
Overall isolated yields and purity of crude products for 14a–e
interfere with the functions of cellular counterparts. RNase H pro-
teins, including human RNase H, are homologous and share a sim-
ilar three-dimensional fold. To address these challenges and to
discover potent RNase H inhibitors we examined the potency of
synthesized modified nucleoside analogs against RNase H and
polymerase function of HIV-1 RT.
No.
Overall yield (%) calcd from 8
Purity of crude products
14a
14b
14c
14d
14e
75
81
82
57
53
87
92
90
78
67
Initially we evaluated the potency of the compounds with mod-
ified phosphates toward the RNase H activity of HIV-1 RT at a fixed
concentration (1 mM) of the compounds. RNase H assay was per-
formed using recombinant RT and RNA/DNA hybrids as previously
2
6–28
b,
ence of 5-(ethylthio)-1H-tetrazole to produce the corresponding
polymer-bound b, -methylenetriphosphitylating reagent 8. The
treatment of unprotected nucleosides (4 mmol, e.g., adenosine
c
-methylenetriphosphitylating reagent (6, 1.2 equiv) in the pres-
described.
The results of the RNase H cleavage are presented
in Figure 1A. Lane W represents the RNase H cleavage products
in the absence of any inhibitor. Lanes a–e represent the RNase H
cleavage products in the presence of the compounds 14a–e,
respectively. These results indicate that the RNase H activity of
HIV-1 RT is inhibited by the cytidine derivative 14d (lane 4). There-
fore, we tested the potency of compound 14d by performing con-
centration-dependent inhibition assays. RNase H inhibition was
c
0
0
(
a), 3 -azido-3 -deoxythymidine (b), thymidine (c), cytidine (d),
and inosine (e)) with 8 (1 mmol) in the presence of 5-(ethylthio)-
H-tetrazole gave 9a–e, respectively. Oxidation with tert-butyl
1
hydroperoxide followed by removal of the cyanoethoxy group with
DBU afforded the corresponding polymer-bound nucleoside b,
not observed up to 100
100 M to 1 mM are presented in Figure 1B. Compound 14d
achieved complete inhibition of the RNase H activity at 700
lM with the compound. The results from
c-methylenetriphosphates 11a–e. Subsequent steps were sup-
l
ported by FT-IR peaks for all the intermediate resins. The cleavage
of polymer-bound compounds was carried out under mild acidic
conditions, DCM/TFA/water/EDT (72.5:23:2.5:2 v/v/v/v). The crude
products had a purity of 67–92% and were purified on the C18 Sep-
lM
(Fig. 1B, lane 6). These results are consistent with those of our
Pak cartridges to afford nucleoside b,c-methylenetriphosphates
14
A
U
B
14d
4
1
4a–e (Scheme 2) in 53–82% overall yield (calculated from 8) (Ta-
W
a b c d e
1
2
3
5 6 7
ble 1) (269–411 mg). The purity of compounds was calculated
based on the weight of pure and crude compounds.
U
Among all the synthesized methylenetriphosphate derivatives,
1
4d is a novel compound. Adenosine, cytidine, and inosine contain
0
0
0
a secondary (2 and/or 3 ) alcohol, a primary (5 ) alcohol, or a free
amino group. Only 5 -O-substituted compounds were purified with
0
high selectivity as a result of this sequence. The presence of the
phosphitylating reagents on the solid support having a hindered
structure allowed for the regioselective reaction of the unprotected
nucleosides with the polymer-bound phosphitylating reagents.
There was no need to protect the free aromatic amino group in
adenosine and cytidine. The most reactive hydroxyl group of
unprotected nucleosides reacted selectively with hindered and
bulky polymer-bound reagents when an excess of nucleoside was
C
C
C
C
C
C
1
13
used. The compounds were characterized by H NMR, C NMR,
3
1
Figure 1. (A) RNase H analysis of HIV-1 RT in the presence of compounds 14a–e.
Lane w represents no inhibitor. The RNase H cleavage of HIV-1 RT was inhibited by
4d; (B) RNase H activity with increased concentration of compounds 14d. Lane 1
(100 M), lane 2 (200 M), lane 3 (300 M), lane 4 (400 M), lane 5 (500 M), lane
6 (700 M), and lane 7 (1 mM). The unhydrolyzed substrate is marked as U,
whereas the cleavage products are marked as C.
P NMR, ESI-TOF, and phosphorus elemental analysis.
Nucleoside methylenetriphosphates remain as attractive probes
1
and non-hydrolyzable triphosphate mimics in studying and inhib-
iting the phosphate transfer in biological systems. The synthesized
compounds were evaluated to determine whether they can inhibit
human immunodeficiency virus type 1 (HIV-1) reverse transcrip-
tase (RT), a critical enzyme in HIV life cycle.
l
l
l
l
l
l
HIV-1 RT converts the single-stranded RNA genome into dou-
ble-stranded DNA that is later integrated into the host genome.
1
4
2
3
w
a
b c d e
Retroviral RTs are multifunctional enzymes possessing DNA poly-
merase activities on both DNA and RNA templates, as well as a
ribonuclease H (RNase H) activity that hydrolyzes the RNA of
RNA/DNA replication intermediates. HIV-1 RT is a heterodimer
consisting of p66 and p51 subunits that share the same sequence
at the N-terminus. The RNase H domain resides in the C-terminus
P+18
24
of p66 subunit, which is not present in p51. Both DNA polymer-
ase and RNase H activities of HIV-1 RT have been considered as po-
tential targets for antiretroviral therapy. However, all currently
used HIV-1 RT inhibitors are directed against the polymerase func-
2
5
tion of the enzyme. Designing compounds that block the RNase H
activity of the HIV-1 RT has been challenging. Although recent
screening efforts led to the discovery of few RNase H inhibitors,
none of them advanced into clinical trials. RNase H has been a chal-
lenging target for a number of reasons. Inhibitors that block the RT-
associated RNase H function have higher cytotoxicity, since they
P
Figure 2. DNA polymerase activity of HIV-1 RT in the presence of compounds 14a–
e (1 mM). Lane w represents no inhibitor. The radiolabeled primer is marked as P,
whereas the fully extended product is marked as P + 18.

Y. Ahmadibeni et al. / Tetrahedron Letters 51 (2010) 3010–3013
3013
earlier studies that showed another modified nucleoside triphos-
References and notes
0
phate derivative, cytidine 5 -O-
inhibited RNase H activity of HIV-1 reverse transcriptase.
a,b-methylene-b-triphosphate
2
2
1. Berkowitz, D. B.; Bose, M.; Pfannenstiel, T. J.; Doukov, T. J. Org. Chem. 2000, 65,
498–4508.
4
We have also tested the potency of these compounds against the
polymerase function of HIV-1 RT (Fig. 2). In comparison with the
wild-type enzyme (lane w), the polymerase activity was not af-
fected in the presence of the compounds 14a–e, (lanes a–e). How-
ever, compound 14c caused the polymerase complex to pause
several positions during DNA synthesis (lane c). Since compound
2.
3.
4.
Jakeman, D. L.; Ivory, A. J.; Blackburn, G. M.; Williamson, M. P. J. Biol. Chem.
2003, 278, 10957–10962.
Lio, X. H.; Brenner, C.; Guranowski, A.; Starzynska, E.; Blackburn, G. M. Angew.
Chem., Int. Ed. 1999, 38, 1244–1247.
Wang, G.; Boyle, N.; Chen, F.; Rajappan, V.; Fagan, P.; Brooks, J. L.; Hurd, T.;
Leeds, J. M.; Rajwanshi, V. K.; Jin, Y.; Prhavc, M.; Bruice, T. W.; Cook, P. D. J. Med.
Chem. 2004, 47, 6902–6913.
5
.
.
Spelta, V.; Mekhalfia, A.; Rejman, D.; Thompson, M.; Blackburn, G. M.; North, R.
A. Br. J. Pharmacol. 2003, 140, 1027–1034.
Hoffenberg, S.; Shannon, T. M.; Noonan, T. P.; Liu, S. B.; Daniel, S.; Fishman, J. B.;
Rubins, J. B.; Misra, H. K.; Wright, G. E.; Dickey, B. F. Mol. Pharmacol. 1996, 49,
1
4d did not inhibit the polymerase activity of HIV-1 RT (lane 4), it
can provide insights for designing of additional compounds, which
may have better inhibitory activity against the RNase H activity.
To the best of our knowledge, this is the first report of the syn-
6
156–164.
7.
Arabshahi, L.; Khan, N. N.; Butler, M.; Noonan, T.; Brown, N. C.; Wright, G. E.
thesis of nucleoside b,c-methylenetriphosphates by using solid-
Biochemistry 1990, 29, 6820–6826.
phase reagents without the need for precursors, such as nucleoside
monophosphates and 5 -methylenediphosphonate analogs. Unre-
8. Taylor, S. D.; Mirzaei, F.; Bearne, S. L. Org. Lett. 2006, 8, 4243–4246.
9. Ono, K.; Nakane, H.; Herdewijn, P.; Balzarini, J.; De Clercq, E. Mol. Pharmacol.
0
1989, 35, 578–583.
acted reagents were removed by extensive washing of the resins
in each step. This solid-phase strategy offered the advantages of
10. Schmitt, L.; Tampe, R. J. Am. Chem. Soc. 1996, 118, 5532–5543.
11. Mohamady, S.; Jakeman, D. L. J. Org. Chem. 2005, 70, 10588–10591.
12. Blackburn, G. M.; England, D. A.; Kolkmann, F. J. Chem. Soc., Chem. Commun.
0
expeditious synthesis, 5 -O-substitution, high selectivity, facile iso-
1981, 930–932.
lation, and purification of final products.
13. Jemielity, J.; Pietrowska-Borek, M.; Starzynska, E.; Kowalska, J.; Stolarski, E.;
Guraniwski, A.; Darzynkiewicz, E. Nucleosides Nucleotides Nucleic Acids 2005,
2
4, 589–593.
Acknowledgments
1
1
4. Kumar, A.; Ye, G.; Ahmadibeni, Y.; Parang, K. J. Org. Chem. 2006, 71, 7915–7918.
5. Parang, K.; Fournier, E. J.-L.; Hindsgaul, O. Org. Lett. 2001, 3, 307–309.
We acknowledge the financial support from National Science
Foundation,GrantNumberCHE0748555.TheworkatMeharryMed-
ical College was supported by NIDA Grant Number R00DA024558.
TheresearchatNCIwassupportedinpartbytheIntramuralResearch
Program of the NIH, NCI, and Center for Cancer Research. We also
acknowledge National Center for Research Resources, NIH, Grant
Number 1 P20 RR16457 for sponsoring the core facility.
16. Parang, K. Bioorg. Med. Chem. Lett. 2002, 12, 1863–1866.
17. Ahmadibeni, Y.; Parang, K. J. Org. Chem. 2005, 70, 1100–1103.
1
1
2
8. Ahmadibeni, Y.; Parang, K. Org. Lett. 2005, 7, 5589–5592.
9. Ahmadibeni, Y.; Parang, K. J. Org. Chem. 2006, 71, 5837–5839.
0. Ahmadibeni, Y.; Parang, K. Angew. Chem., Int. Ed. 2007, 46, 4739–4743.
21. Ahmadibeni, Y.; Parang, K. Org. Lett. 2007, 9, 4483–4486.
22. Ahmadibeni, Y.; Dash, C.; Hanley, M. J.; Le Grice, S. F. J.; Agarwal, H. K.; Parang,
K. Org. Biomol. Chem. 2010, 8, 1271–1274.
23. Telesnitsky, A.; Goff, S. P. In Retroviruses; Coffin, J. M., Hughes, S. H., Varmus, H.
E., Eds.; Cold Spring Harbor Laboratory Press: Plainview, New York, 1997; pp
1
21–160.
Supplementary data
2
4. Le Grice, S. F. In Reverse Transcriptase; Skalka, A. M., Goff, S., Eds.; Cold Spring
Harbor Laboratory Press: Plainview, New York, 1993; pp 163–192.
Supplementary data (experimental procedures and character-
ization of resins with IR and final compounds with NMR, high-res-
olution mass spectrometry, and quantitative phosphorus analysis,
enzyme assay) associated with this article can be found, in the on-
line version, at doi:10.1016/j.tetlet.2010.04.005.
25. DeClercq, E. J. Clin. Virol. 2004, 30, 115–133.
2
6. Le Grice, S. F.; Cameron, C. E.; Benkovic, S. J. Methods Enzymol. 1995, 262, 130–
44.
7. Dash, C.; Rausch, J. W.; Le Grice, S. F. J. Nucleic Acids Res. 2004, 32, 1539–1547.
1
2
28. Dash, C.; Fisher, T. R.; Prasad, V. R.; Le Grice, S. J. Biol. Chem. 2006, 281, 27873–
7881.
2