Synthesis of AZTpSpCX2ppSA and AZTp SpCX2ppSAZT: Hydrolysis-resistant potential inhibitors of the AZT excision reaction of HIV-1 RT

Article abstract of DOI:10.1021/ol7023746

(Chemical Equation Presented) We report an efficient, one-flask route for synthesis of AZTp_Sp_CX2pp_SA and AZTp _Sp_CX2pp_SAZT, where X = H and X = F. This route makes use of the differential susceptibility to oxidation of H-phosphonate mono- and diesters, to allow a series of sequential reactions without requiring isolation of intermediates. These compounds are hydrolysis-resistant versions of the AZTppppA that results from excision of AZT by AZT-resistant HIV reverse transcriptase (RT). This family of compounds may therefore be useful in further study of the AZT excision reaction, as well as in drug design.

Full text of DOI:10.1021/ol7023746

ORGANIC
LETTERS
2007
Vol. 9, No. 25
5243-5246
Synthesis of AZTpSpCX2ppSA and
AZTpSpCX2ppSAZT: Hydrolysis-Resistant
Potential Inhibitors of the AZT Excision
Reaction of HIV-1 RT
Qianwei Han,† Stefan G. Sarafianos,†,‡,¶ Eddy Arnold,†,‡ Michael A. Parniak,§
Barbara L. Gaffney,† and Roger A. Jones*,†
Department of Chemistry and Chemical Biology, Rutgers, The State UniVersity of New
Jersey, 610 Taylor Road, Piscataway, New Jersey 08854, Center for AdVanced
Biotechnology and Medicine, Piscataway, New Jersey 08854, and Department of
Molecular Genetics & Biochemistry, UniVersity of Pittsburgh School of Medicine,
Pittsburgh, PennsylVania 15261
r.jones@rutgers.edu
Received October 3, 2007
ABSTRACT
We report an efficient, one-flask route for synthesis of AZTpSpCX2ppSA and AZTpSpCX2ppSAZT, where X
)
H and X
)
F. This route makes use
of the differential susceptibility to oxidation of H-phosphonate mono- and diesters, to allow a series of sequential reactions without requiring
isolation of intermediates. These compounds are hydrolysis-resistant versions of the AZTppppA that results from excision of AZT by AZT-
resistant HIV reverse transcriptase (RT). This family of compounds may therefore be useful in further study of the AZT excision reaction, as
well as in drug design.
HIV-1 RT (human immunodeficiency virus type 1 reverse
transcriptase) synthesizes DNA from the viral RNA strand
and is a major target for anti-retroviral agents.1 Nucleoside
RT inhibitors (NRTIs), such as 3′-azido-3′-deoxythymidine
(AZT), function as chain terminators and are used in clinical
treatment of HIV infections.2 Unfortunately, resistance to
such drugs commonly develops as the virus mutates. The
principal mechanism for this resistance is enhanced removal
of the chain-terminating nucleotide from the blocked DNA
strand by an RT-catalyzed reaction that uses adenosine
triphosphate (ATP) as a nucleophile.3 This excision unblocks
the DNA, permitting elongation to resume. AZT is particu-
larly susceptible to this excision reaction because steric
constraints from the azido group keep it in the nucleotide
binding site of the catalytic center, where excision is favored.4
† Rutgers.
‡ Center for Advanced Biotechnology and Medicine.
§ University of Pittsburgh School of Medicine.
¶ Current address: Department of Molecular Microbiology & Immunol-
ogy, University of Missouri School of Medicine, 1201 E. Rollins Dr.,
Columbia, MO 65211.
(2) (a) Parniak, M. A.; Sluis-Cremer, N. AdV. Pharmacol. 2000, 49, 67-
109. (b) Gallant, J. E.; Gerondelis, P. Z.; Wainberg, M. A.; Shulman, N.
S.; Haubrich, R. H.; Clair, M. S.; Lanier, E. R.; Hellmann, N. S.; Richman,
D. D. AntiViral Ther. 2003, 8, 489-506. (c) DeClercq, E. Pure Appl. Chem.
1998, 70, 567-577.
(1) Telesnitsky, A.; Goff, S. P. In RetroViruses; Coffin, J. M., Hughes,
S. H., Varmus, H. E., Eds.; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, NY, 1997.
10.1021/ol7023746 CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/08/2007

Scheme 1
The dinucleoside tetraphosphate that results from excision
bisphosphonates are isosteric with phosphates, only CF2
analogues are isopolar9 and may enhance critical interactions
at the binding site.
of AZT is 5′-5′′′-AZTp4A, which has recently been found
to be an excellent chain-terminating substrate for resistant
RT.5 Hydrolysis-resistant versions of AZTp4A containing two
outer thiophosphates and a central bisphosphonate, such as
AZTpSpCX2ppSA, X ) H (6) and X ) F (7), and the related
compounds with AZT at both ends, AZTpSpCX2ppSAZT, X
) H (8) and X ) F (9), may therefore serve as useful
inhibitors of this excision reaction, thereby reducing AZT
resistance.6
Thiophosphates as well as bisphosphonates are known to
undergo reduced hydrolysis by cellular enzymes.7,8 Therefore,
these compounds should be resistant to such hydrolysis at
all four phosphorus atoms and may lead to particularly
promising candidates for long-lasting inhibitors of the AZT
excision reaction. The adenosine in 6/7 may play a favorable
role in tight binding to mutated forms of RT, while the
presence of AZT at both ends of 8/9 may promote binding
to wild type RT. Further, although both CH2 and CF2
As part of our ongoing efforts to develop improved
synthetic methods for dinucleoside polyphosphates and their
analogues, we now report an efficient, one-flask synthesis
of 6-9. This route makes use of the ability of H-phosphonate
diesters to undergo modification at the phosphorus atom,
along with the differential susceptibility to oxidation of
H-phosphonate mono- and diesters, to allow the series of
sequential reactions shown in Scheme 1, without requiring
isolation of intermediates. Because H-phosphonate mo-
noesters are not easily oxidized, we can use 5a/b, despite
the presence of excess S8 from the previous step, to condense
with modified trimetaphosphates 4a/b. It is only after the
condensation has taken place that the excess S8 in the reaction
mixture then converts the product H-phosphonate diesters
(not shown) to the dithiophosphates 6-9.
The thiotrimetaphosphates 4a/b were prepared by a route
we recently reported for the synthesis of unmodified di-
nucleoside tetraphosphates.10 Knorre some time ago devel-
oped a synthetic procedure for preparation of adenosine
trimetaphosphate using a carbodiimide,11 and this useful
intermediate has been used by others to prepare a variety of
nucleotide derivatives.12 Eckstein introduced a particularly
convenient method for preparing nucleoside trimetaphos-
(3) (a) Meyer, P. R.; Matsuura, S. E.; So, A. G.; Scott, W. A. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 13471-13476. (b) Meyer, P. R.; Matsuura,
S. E.; Mian, A. M.; So, A. G.; Scott, W. A. Mol. Cell 1999, 4, 35-43. (c)
Meyer, P. R.; Smith, A. J.; Matsuura, S. E.; Scott, W. A. Antimicrob. Agents
Chemother. 2006, 50, 3607-3614.
(4) (a) Boyer, P. L.; Sarafianos, S. G.; Arnold, E.; Hughes, S. H. J. Virol.
2001, 75, 4832-4842. (b) Boyer, P. L.; Sarafianos, S. G.; Arnold, E.;
Hughes, S. H. J. Virol. 2002, 76, 9143-9151.
(5) Dharmasena, S.; Pongracz, Z.; Arnold, E.; Sarafianos, S. G.; Parniak,
M. A. Biochemistry 2007, 46, 828-836.
(6) Sarafianos, S. G.; Hughes, S. H.; Arnold, E. Int. J. Biochem. Cell
Biol. 2004, 36, 1706-1715.
(9) (a) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. J. Chem. Soc., Perkin
Trans. 1 1984, 1119-1125. (b) Blackburn, G. M.; Eckstein, F.; Kent, D.
E.; Perree, T. D. Nucleosides Nucleotides 1985, 4, 165-167.
(10) Han, Q.; Gaffney, B. L.; Jones, R. A. Org. Lett. 2006, 8, 2075-
2077.
(11) Knorre, D. G.; Kwibatoo, V. A.; Samukov, V. V. FEBS Lett. 1976,
70, 105-108.
(7) (a) Eckstein, F. Annu. ReV. Biochem. 1985, 54, 367-402. (b) Eckstein,
F. Biochemie 2002, 84, 841-848. (c) McLennan, A. G.; Taylor, G. E.;
Prescott, M.; Blackburn, G. M. Biochemistry 1989, 28, 3868-3875.
(8) Blackburn, G. M.; Taylor, G. E.; Thatcher, G. R. J.; Prescott, M.;
McLennan, A. G. Nucleic Acids Res. 1987, 15, 6991-7004.
5244
Org. Lett., Vol. 9, No. 25, 2007

Scheme 2
phates and R-thionucleoside triphosphates using 2-chloro-
sulfurization. We are able to separate all diastereomeric
products, four each for unsymmetrical 6/7 and three each
for symmetrical 8/9 (for which RS and SR are equivalent),
with total isolated yields ranging from 57 to 82%. The related
compounds ApSpCH2ppSA8 and AppCH2ppA20 have previously
been synthesized as hydrolysis-resistant versions of the
signaling molecule Ap4A, but in modest yields.
4H-1,3,2-benzodioxaphosphorin-4-one, followed by oxida-
tion or sulfurization, respectively.13 The procedure was
further extended to prepare nucleoside triphosphates with
various modifications14 as well as with substituted âγ-
methylenes along with R-P-thioates.15
The first step in our procedure is reaction of a nucleoside,
in this case AZT (1), with 2-chloro-4H-1,3,2-benzodioxa-
phosphorin-4-one in DMF to give 2. We then react 2 with a
pyrophosphate analogue,16 either methylene or difluoro-
methylene17 diphosphonate, to form the modified trimeta-
phosphates, 3a or b, respectively, followed by addition of
excess S8 to give 4a/b.
We have assigned the configurations of the diastereomers
of 6-9 based on their relative susceptibility to hydrolysis
by snake venom phosphodiesterase. Eckstein demonstrated
that the R diastereomer of pppSA was cleaved significantly
faster than the S diastereomer.21 Further, he found that the
R diastereomer had a longer retention time on reverse phase
HPLC. Blackburn and McLennan used this selective enzy-
matic hydrolysis approach to assign configurations of the
three diastereomers of ApSpCH2ppSA.8 We have digested 0.94
mM samples of each diastereomer of 6-9 with 0.2-5 mg
of snake venom phosphodiesterase and plotted the %
undigested 6-9 remaining, determined by HPLC, as a
function of time. The results for AZTp4 p3CH2p2p1 A (6a-
The 5′-H-phosphonates of either adenosine, 5a, or AZT,
5b, were made using 2-chloro-4H-1,3,2-benzodioxaphos-
phorin-4-one.18 To prepare adenosine H-phosphonate, 5a, we
first protect adenosine, 10, using N,N-dimethylformamide
dimethyl acetal to give 11 (Scheme 2).19 This transient pro-
tection is conveniently reversed after the phosphitylation by
overnight hydrolysis in dilute aqueous ammonia. The product
is then converted to the tri-n-butylammonium form using
cation exchange resin and dried so as to form a DMF solution
that can be employed directly in the reaction with 4a/b. AZT
H-phosphonate, 5b, does not require prior protection or
conversion to the alkylammonium form. After the condensa-
tion, which is catalyzed by ZnCl2, the second sulfurization
is effected immediately by the excess S8 left from the first
S
S
d) are shown in Figure 1A and demonstrate that the
(12) (a) Imai, J.; Torrence, P. F. Biochemistry 1984, 23, 766-774. (b)
Kappler, F.; Hampton, A. In Nucleic Acid Chemistry; Townsend, L. B.,
Tipson, R. S., Eds.; John Wiley & Sons: New York, 1986; Vol. 3. (c)
Puri, N.; Hunsch, S.; Sund, C.; Ugi, I.; Chattopadhyaya, J. Tetrahedron
1995, 51, 2991-3014. (d) Sood, A.; Kumar, S.; Nampalli, S.; Nelson, J.
R.; Macklin, J.; Fuller, C. W. J. Am. Chem. Soc. 2005, 127, 2394-2395.
(13) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631-635.
(14) Li, P.; Xu, Z.; Liu, H.; Wennefors, C. K.; Dobrikov, M. I.; Ludwig,
J.; Shaw, B. R. J. Am. Chem. Soc. 2005, 127, 16782-16783.
(15) Boyle, N. A.; Fagan, P.; Brooks, J. L.; Prhavc, M.; Lambert, J.;
Cook, P. D. Nucleosides Nucleotides Nucleic Acids 2005, 24, 1651-1664.
(16) (a) 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. (b) Boyle, N. A.;
Rajwanshi, V. K.; Prhavc, M.; Wang, G.; Fagan, P.; Chen, F.; Ewing, G.
J.; Brooks, J. L.; Hurd, T.; Leeds, J. M.; Bruice, T. W.; Cook, P. D. J.
Med. Chem. 2005, 48, 2695-2700. (c) Mohamady, S.; Jakeman, D. L. J.
Org. Chem. 2005, 70, 10588-10591.
Figure 1. Plots of % undigested tetraphosphates during enzymatic
degradation for the diastereomers of AZTp4 p3CH2p2p1 A, 6a-d
(17) Boyle, N. A. Org. Lett. 2006, 8, 187-189.
S
S
(18) Marugg, J. E.; Tromp, M.; Kuyl-Yeheskiely, E.; van der Marel, G.
A.; van Boom, J. H. Tetrahedron Lett. 1986, 27, 2661-2664.
(19) Zemlicka, J. Collect. Czech. Chem. Commun. 1963, 28, 1060-1062.
(panel A), and AZTp4 p3CF2p2p1 A, 7a-d (panel B).
S
S
Org. Lett., Vol. 9, No. 25, 2007
5245

diastereomer with the longest retention time (6d) is cleaved
faster than the others (26% left after 1 h), consistent with an
RP1,RP4 assignment. The diastereomer with the shortest
retention time (6a) exhibits almost no cleavage, even after
7 h, consistent with an SP1,SP4 assignment. Of the two
remaining diastereomers with intermediate retention times,
one (6c) is cleaved significantly faster than the other, with
46% left after 1 h. Because its cleavage produced almost
entirely adenosine thiomonophosphate and adenosine (Sup-
porting Information), we assign it RP1,SP4. The other inter-
mediate diastereomer (6b) showed 93% left after 1 h and
produced almost entirely AZT thiomonophosphate, consistent
with an assignment of SP1,RP4.
For comparison, we have also synthesized the unmodified
tetraphosphates, AZTp4A (12) and AZTp4AZT (13), and two
analogues with central bisphosphonates but no thiophos-
phates, AZTppCH2ppA (14) and AZTppCF2ppA (15).22 They
show much faster degradation with snake venom phosphodi-
esterase than 6-9, with only 50% left after about 1 min
(Supporting Information).
This synthetic approach allows convenient one-flask
synthesis of dinucleoside tetraphosphate analogues. The
particular thiophosphate/bisphosphonate analogues described
here are hydrolyzed by the well-known phosphodiesterase
much more slowly than unmodified tetraphosphates, par-
ticularly their SS diastereomers. This family of compounds
may therefore be useful in further study of the AZT excision
reaction, as well as in drug design.
Comparable degradation of AZTp4 p3CF2p2p1 A (7) re-
S
S
quired 2.5 times as much phosphodiesterase, and the results
are shown in Figure 1B with corresponding assignments.
Similar plots and assignments for 8/9 were done in the same
fashion and are shown in the Supporting Information. In all
four sets of diastereomers, the order of retention time on
reverse phase chromatography correlates with the rate of
enzyme degradation, with the compound showing the longest
retention time degrading the fastest. The 31P NMR chemical
shifts of all the diastereomers of 6-9 display extremely small
differences, with no obvious correlation with configuration.
Acknowledgment. This work was supported by NIH
grants GM066671 and GM079760.
Supporting Information Available: Synthetic methods,
1
spectra (UV, MS, H, 13C, 19F, and 31P NMR), degradation
methods, and plots of enzymatic degradation versus time.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL7023746
(20) Guranowski, A.; Biryukov, A.; Tarussova, N. B.; Khomutov, R.
M.; Jakubowski, H. Biochemistry 1987, 26, 3425-3429.
(21) Burgers, P. M. J.; Eckstein, F. Proc. Natl. Acad. Sci. U.S.A. 1978,
75, 4798-4800.
(22) AZTp4A, AZTp4AZT, and AZTppCX2ppAZT (X ) H, F) were all
made by our previously reported trimetaphosphate method using a nucleo-
side monophosphate.10 AZTp4AZT was also made by the H-phosphonate
method reported here and gave comparable yields.
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Org. Lett., Vol. 9, No. 25, 2007