Synthesis of prodrug candidates: conjugates of amino acid with nucleoside boranophosphate.

Article abstract of DOI:10.1021/ol025832b

[structure: see text] Preparation of antiviral and anticancer prodrug candidates, P-tyrosinyl(P-O)-5'-P-nucleosidyl boranophosphates, is described. One-pot synthesis via a phosphoramidite method resulted in the title compounds with good yields. The P-boranophosphate diastereomers were separated by RP-HPLC, and their structures were confirmed by 1H and 31P NMR spectroscopy and MS analysis.

Full text of DOI:10.1021/ol025832b

ORGANIC
LETTERS
2002
Vol. 4, No. 12
2009-2012
Synthesis of Prodrug Candidates:
Conjugates of Amino Acid with
Nucleoside Boranophosphate
Ping Li and Barbara Ramsay Shaw*
Department of Chemistry, Box 90346, Duke UniVersity,
Durham, North Carolina 27708-0346
brshaw@chem.duke.edu
Received March 6, 2002
ABSTRACT
Preparation of antiviral and anticancer prodrug candidates, P-tyrosinyl(P-O)-5′-P-nucleosidyl boranophosphates, is described. One-pot
synthesis via a phosphoramidite method resulted in the title compounds with good yields. The P-boranophosphate diastereomers were
separated by RP-HPLC, and their structures were confirmed by 1H and 31P NMR spectroscopy and MS analysis.
The design of many antiviral drugs focuses on the inhibition
of viral polymerases and reverse transcriptases (RT),1 the
key enzymes in the replicative cycle of any virus. Most of
the available drugs are nucleoside analogues,2 for example,
3′-azido-3′-deoxythymidine (AZT, Zidovudine),2c the first of
five anti-HIV nucleoside analogues2 approved by the FDA
to treat AIDS patients. The activation mechanism of anti-
HIV nucleoside analogues in vivo2f,3 involves phosphoryl-
ation by cellular kinases into the corresponding 2′-deoxy-
nucleoside mono- (dNMP), di- (dNDP), and triphosphate
(dNTP). Then the biologically active dNTP is incorporated
into the growing viral DNA chain, causing chain termination.
Modified nucleosides are also used in cancer therapy. For
example, 5-fluoro-2′-deoxyuridine (FdU) exerts its effects
through formation of the nucleotide analogue 5-fluoro-2′-
deoxyuridine monophosphate (FdUMP), which blocks DNA
biosynthesis by deactivating thymidylate synthase (TS), a
key enzyme for the production of thymidine-5′-monophos-
phate (TMP) from deoxyuridine-5′-monophosphate (dUMP).4,5
However, in many cases, the nucleoside analogue itself is
a poor substrate for cellular kinases needed for its activation.6
In some cases, activation of the particular nucleoside
analogue may be restricted in cells where the nucleoside
kinase activity is low or even deficient.7 Therefore, several
(1) (a) Kohl, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J.; Heimbach,
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10.1021/ol025832b CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/21/2002

approaches have been advanced to circumvent the initial
kinase-catalyzed phosphorylation step. The so-called ‘‘pro-
drug approach” suggests the use of nucleoside analogues in
an already phosphorylated form, masked with various
protecting groups. These prodrugs are designed to readily
penetrate into cells and easily convert intracellularly into
dNMPs.3,8
We propose to use boranophosphate diesters, in which one
of the nonbridging oxygen atoms of a phosphate diester
group is replaced by a borane group (BH3),9 as a candidate
for such a prodrug. As reviewed by Shaw,9f,g boranophos-
phates are more lipophilic and nuclease-resistant than the
normal phosphate diesters.9e,10a Our recent studies indicate
that the RP isomers of R-P-boronated dNTPs are better
substrates than natural dNTPs for viral DNA polymerases,11
making them more selective for incorporation into viral
DNA. Meyer et al.12 showed that the R-(RP)boranodiphos-
phate of AZT is a 10-fold better substrate for diphosphate
kinase than normal AZT diphosphate. Moreover, the R-(RP)-
boranotriphosphate of AZT has a 9-fold increased efficiency
with HIV-RT compared with normal AZT triphosphate
(AZTTP). The R-P-borano derivative of AZTTP has in-
creased stability toward repair mechanisms that contribute
to HIV drug resistance,12 possibly because an R-P-borono-
phosphate in DNA is more resistant to nuclease than normal
phosphate.9e Finally, boronated nucleotides may offer a
unique advantage over other modified congeners because
they could be used for boron neutron capture therapy
(BNCT),13 a radiation therapy that can selectively destroy
cells that have preferentially taken up boron. All of these
unique properties of boranophosphates make them promising
candidates for design of antiviral and anticancer prodrugs.
As a prototypic masking group, we have chosen L-tyrosine.
Amino acids are known as good carriers for the nucleoside
prodrugs, forming nontoxic hydrolysis products.8b,14 The
phenyl ester bond, present in tyrosine-containing conjugates,
is reported to be the most labile bond among the phenyl-,
benzyl-, and alkylphosphates15 because the negative charge
formed upon hydrolysis can be delocalized into the aromatic
ring. It facilitates the chemical or enzymatic hydrolysis of
the prodrugs to form the biologically active nucleotide
derivatives.
Specifically, in this article we report the first synthesis of
P-tyrosinyl(P-O)-5′-P-nucleosidyl boranophosphates 5.
Our first attempt to prepare the conjugate was based on
an H-phosphonate approach that we generally use for
synthesis of various nucleoside boranophosphate deriva-
tives.10 Condensation of uridine H-phosphonate 1 with
protected tyrosine 3 gave the compounds 4 in only 25-30%
yield after isolation (Scheme 1). Instability of the phenyl
H-phosphonate diester during silica gel chromatography
could be a reason for the low yield. Although subsequent
(4) (a) Tong, Y.; Liu-Chen, X.; Erickan-Abali, E. A.; Capiaux, G. M.;
Zhao, S.; Banedee, D.; Bertino, J. R. J. Biol. Chem. 1998, 273, 11611. (b)
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M. E.; Forsthoefel, A. M.; Barbour, K. W.; Spencer, H. T.; Berger, F. G.
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Scheme 1
(8) (a) Groschel, B.; Cinatl, J.; Perigaud, C.; Gosselin, G.; Imbach, J.
L.; Doerr, H. W.; Cinatl, J. AntiViral Res. 2002, 53, 143. (b) Shafiee, M.;
Deferme, S.; Villard, A. L.; Egron, D.; Gosselin, G.; Imbach, J. L.; Lioux,
T.; Pompon, A.; Varray, S.; Aubertin, A. M.; Van den Mooter, G.; Kinget,
R.; Perigaud, C.; Augustijns, P. J. Pharm. Sci. 2001, 90, 448. (c) Perigaud,
C.; Gosselin, G.; Lefebver, I.; Girardet, J. L.; Benzaria, S.; Barber, I.;
Imbach, J. L. Bioorg. Med. Chem. Lett. 1993, 3, 2521. (d) Jones, R. J.;
Bischofberger, W. AntiViral Res. 1995, 27, 1. (e) Meier, C. Synlett 1998,
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(9) (a) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J. Am. Chem. Soc. 1990,
112, 9000. (b) Tomasz, J.; Shaw, B. R.; Porter, K.; Spielvogel, B. F.; Sood,
A. Angew. Chem., Int. Ed. Engl. 1992, 31, 1373. (c) Shaw, B. R.; Madison,
J.; Sood, A.; Spielvogel, B. F. Methods Mol. Biol. 1993, 20, 225. (d) Li,
H.; Hardin, C.; Shaw, B. R. J. Am. Chem. Soc. 1996, 118, 6606. (e) Porter,
K. W.; Briley, J. D.; Shaw, B. R. Nucleic Acids Res. 1997, 25, 1611. (f)
Shaw, B. R.; Sergueev, D.; He. K.; Porter. K.; Summers, J.; Sergueeva, Z.;
Rait, V. Method Enzymol. 2000, 313, 226. (g) Summers, J. S.; Shaw, B. R.
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(11) Unpublished results.
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2010
Org. Lett., Vol. 4, No. 12, 2002

silylation and boronation proceeded smoothly, the overall
yield of the final product 5a after deprotection was less than
10%. Reaction of uridine 5′-boranomonophosphate (5′-
UMPB) 2 with protected tyrosine 3 using DCC as condensing
agent was also unsuccessful. Finally, we decided to synthe-
size the boranophosphate conjugate through a phosphor-
amidite intermediate obtained from protected tyrosine.
Scheme 2a
One of the crucial points in our synthesis was the choice
of the protecting groups for the amine and acid functionalities
of L-tyrosine. Conditions for cleaving these protecting groups
have to fulfill the stability requirements for the final
conjugates, which carry base- and nucleophile-sensitive
functionalities. Moreover, at least one UV-active aryl group
was preferred because it could produce a tag for monitoring
the reaction and chromatography. Therefore, the tert-butyl-
oxycarbonyl (t-Boc) and p-nitrobenzyl groups were selected
as amino and acid protections, respectively.
The syntheses of the desired P-tyrosinyl(P-O)-5′-P-nucleo-
sidyl boranophosphates 5a-c were carried out as outlined
in Scheme 2. Tyrosine was additionally protected with a
p-nitrobenzyl group, as described previously,16 and the
resulting compound 3 was phosphorylated by 2-cyanoethyl
N,N,N′,N′-tetraisopropylphosphane (2-OCEt-P[N(iPr2)2]) in
the presence of 1H-tetrazole to give phosphoramidite 7. After
40 min, nucleoside and another portion of 1H-tetrazole were
added into the reaction mixture. Condensation was completed
in 15 min as evidenced by 31P NMR spectra, where the
singlet at 148 ppm for phosphoramidite 7 was transformed
into two singlets around 135 ppm corresponding to the
diastereomers of phosphotriester conjugates 8 (ratio 1:1).
Without purification, in situ boronation of intermediates 8
resulted in the formation of boranophosphotriesters 9. The
presence of the P f B bond in intermediates 9 was confirmed
by 31P NMR spectra, which showed the characteristic broad
peak centered at 116 ppm.10 Of several borane complexes
used for boronation, borane-dimethyl sulfide (2.5 equiv at 0
°C for 45 min) gave the best results. The reaction mixture
was then evaporated to dryness and extracted with ethyl
acetate and water. The organic layers were concentrated and
treated with a mixture of ammonium hydroxide and methanol
(1:1, v/v) for 10 h. The removal of the t-Boc group was
carried out by treating compounds 10 with trifluoroacetic
acid in acetonitrile (1:1, v/v) for 25 min. The final products
5 were precipitated by anhydrous ethyl ether and isolated
by ion-exchange chromatography. The overall yields (from
3 to 5a-c) were 37-46%. The conjugates, P-tyrosinyl(P-
O)-5′-P-nucleosidyl boranophosphates, were identified by 31P
a (i) p-Nitrobenzyl bromide; (ii) 2-OCEt-P[N(iPr2)2], tetrazole,
HN(iPr2)2; (iii) nucleoside, tetrazole; (iv) BH3‚SMe2; (v) NH4OH,
MeOH (1:1, v/v); (vi) TFA in CH3CN (1:1, v/v).
each conjugate were separated by reverse-phase HPLC (RP-
HPLC) using gradient elution for 5a,b and isocratic elution
for 5c.
1
NMR (typical broad peaks at 91 ppm), H NMR spectros-
copy, and HRMS. Two P-boranophosphate diastereomers for
To summarize, we have synthesized the first nucleoside-
amino acid conjugates connected through a boranophosphate
linkage. The one-pot reaction gave the products 5a-c in 37-
46% overall yield after isolation. The P-boranophosphate
diastereomers of uridine, 5-FdU, and AZT were separated
by RP-HPLC. Such conjugates are expected to show good
bioavailability and enhanced cellular uptake. They should
be hydrolyzed enzymatically, leaving an amino acid and
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E.; Balzarini, J. AntiViral Chem. Chemother. 2000, 11, 111. (b) Chang, S.;
Griesgraber, G. W.; Southern, P. J.; Wagner, C. R. J. Med. Chem. 2001,
44, 223.
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Chem. 1981, 24, 479. (b) Farrow, S. N.; Jones, A. S.; Kumar, A.; Walker,
R. T.; Balzarini, J.; De Clercq, E. J. Med. Chem. 1990, 33, 1400.
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Nguyen, A. C.; Kenyon, G. L. J. Am. Chem. Soc. 1987, 109, 5343.
Org. Lett., Vol. 4, No. 12, 2002
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putatively nontoxic boronated nucleotide.17 The improved
substrate properties, increased lipophilicity, and nuclease
resistance imparted by the borane group, in conjunction with
the potential utility as a carrier of 10B in boron neutron
capture therapy, make the P-tyrosinyl(P-O)-5′-P-nucleosidyl
boranophosphate a promising candidate for antiviral and
anticancer prodrugs.
Acknowledgment. This work was supported by NIH
grant R01 GM57693 to B.R.S. We thank Drs. Zinaida
Sergueeva, Dmitri Sergueev, and Mikhail Dobrikov for
helpful discussion and critical comments.
Supporting Information Available: Spectra data for new
compounds 5. This material is available free of charge via
the Internet at http://pubs.acs.org.
(17) Hall, I. H.; Burnham, B. S.; Rajendran, K. G.; Chen, S. Y.; Sood,
A.; Spielvogel, B. F.; Shaw, B. R. Biomed. Pharmacother. 1993, 47, 79.
OL025832B
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Org. Lett., Vol. 4, No. 12, 2002