"Lock-in" modified cycloSal nucleotides - The second generation of cycloSal prodrugs

Article abstract of DOI:10.1081/NCN-200059298

A new generation of cycloSal-pronucleotides is presented. CycloSal-d4TMPs have been modified by introduction of an esterase-cleavable site in order to trap them inside cells. Hydrolysis studies in different media (PBS, CEM/0- and liver extracts) and anti-

Full text of DOI:10.1081/NCN-200059298

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“LOCK-IN” MODIFIED CYCLOSAL NUCLEOTIDES—THE
SECOND GENERATION OF CYCLOSAL PRODRUGS
a
D. Vukadinović , N. P. H. Böge a , J. Balzarini b & C. Meier a
a Institute of Organic Chemistry, University of Hamburg , Hamburg, Germany
b Rega Institute for Medical Research, Katholieke Universiteit , Leuven, Belgium
Published online: 15 Nov 2011.
To cite this article: D. Vukadinović , N. P. H. Böge , J. Balzarini & C. Meier (2005) “LOCK-IN” MODIFIED CYCLOSAL
NUCLEOTIDES—THE SECOND GENERATION OF CYCLOSAL PRODRUGS, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7,
939-942, DOI: 10.1081/NCN-200059298
To link to this article: http://dx.doi.org/10.1081/NCN-200059298
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):939–942, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059298
‘‘LOCK-IN’’ MODIFIED CYCLOSAL NUCLEOTIDES—THE SECOND
GENERATION OF CYCLOSAL PRODRUGS
5
D. Vukadinovi´c and N. P. H. B¨oge
Institute of Organic Chemistry, University of
Hamburg, Hamburg, Germany
5
J. Balzarini
Rega Institute for Medical Research, Katholieke Universiteit, Leuven,
Institute of Organic Chemistry, University of Hamburg, Hamburg, Germany
Belgium
5
C. Meier
5
A new generation of cycloSal-pronucleotides is presented. CycloSal-d4TMPs have been modified by
introduction of an esterase-cleavable site in order to trap them inside cells. Hydrolysis studies in
different media (PBS, CEM/0- and liver extracts) and anti-HIV evaluation of separated dia-
stereomers revealed unexpected differences between the isomers.
Keywords Prodrugs, Pronucleotides, CycloSal, Antiviral Activity
INTRODUCTION
The cycloSal prodrugs have been developed as an intracellular delivery system
for therapeutically active nucleotides and have already been applied to different
nucleoside analogues leading to improved biological activities.[1] However, due to
the lipophilic nature of the cycloSal pronucleotides, it cannot be excluded that a
concentration equilibrium is formed through the membrane. Therefore, the
intention was to modify the phosphate triesters in order to trap them inside cells. To
achieve this goal, triesters bearing esterase-cleavable sites in the cycloSal-moiety
were synthesized. Ester groups were introduced into the 3-position of the mask-
ing unit which should lead to the much more polar cycloSal-NMP-carboxylate
or -alcohol after intracellular enzymatic cleavage. The increased polarity should
inhibit the efflux and so achieve the intracellular ‘‘lock-in’’ of the prodrug.[2]
Address correspondence to C. Meier, Institute of Organic Chemistry, University of Hamburg, Martin-Luther-
King-Platz 6, Hamburg D-20146, Germany.
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D. Vukadinovic´ et al.
RESULTS
In order to use the synthetic approach that had been used previously for the
synthesis of the cycloSal prodrugs, the appropriate ester-group bearing salicyl alcohols
were needed.[3] For the synthesis of the 3-AcEt- and 3-LevEt-phenols transesterfica-
tion reactions of ethyl- or levulinlylacetate and 2-[2-hydroxyphenyl]-ethanol were
carried out.[4] Methanolysis of dihydrocoumarine yielded 3-MePr phenol (Figure 1).
Esterfication of 3-[2-hydroxyphenyl]-propionic acid with N,N-dimethylforma-
mide-neopentylacetale and t-BuOH gave the desired 3-t-BuPr phenol.[5] These ester-
modified phenol derivatives were hydroxymethylated following our previously
reported protocol to yield the corresponding salicyl alcohols.[3] These diols were
reacted with PCl3 to yield cyclic chlorophosphanes which were then used as
phosphitylation agents of d4T. The resulting phosphites were oxidized to give the
cycloSal-triesters 1--6. The reaction of 3-t-BuPr-cycloSal-d4TMP 3 with TFA led to
3-HPr-cycloSal-d4TMP 1 and the levulinyl group of 3-LevEt-cycloSal-d4TMP 6 was
cleaved by hydrazine Á hydrate to yield 3-OHEt-cycloSal-d4TMP 4. Separation of
the diastereomers of 3-MePr- (2) and 3-AcEt-cycloSal-d4TMP 5 by preparative RP-
HPLC gave four isomers 2 fast, 2 slow and 5 fast, 5 slow, respectively. They
were analysed according to their hydrolysis half-lives in different media (PBS, CEM/
0- and liver extracts), their anti-HIV activities.
The triester 2 and 5 were studied concerning their stability in 25 mM aqueous
phosphate buffer (PBS) at pH 7.3. The half-lives t1/2 are summarized in Table 1. The
obtained stability values for compounds 5 clearly revealed differences between t1/2
for the cycloSal-isomer 5 fast and 5 slow and the corresponding diastereomic
mixture 5. Triester 5 slow isomer (18.1 h) was approximately twice as stable as its 5
fast isomer (7.9 h) while the mixture of 5 fast and 5 slow showed a t1/2 of 13.6 h.
Obviously, the configuration at the phosphorus atom had a great influence on
hydrolysis half-lives. As expected the diastereomeric mixture 3-OHEt-cycloSal-d4T 4
showed the same chemical stability as the mixtures of esters 5. The t1/2 values
FIGURE 1 Scheme of the ‘‘lock-in’’ concept and nomenclature of the cycloSal-d4TMPs.

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D. Vukadinovic´ et al.
measured for 2 showed the same effect as observed for diastereomers 5. The 2
slow (16.7 h) was approximately twice as stable as 2 fast (8.1 h) while for the
diastereomeric mixture of 2 a t1/2 value of 12.4 h was measured. The chemical
stability of the acid 3-HPr-cycloSal-d4T 1 (20.4 h) was 1.3- to 2.8-fold higher compared
to the esters. This increased stability may be attributed to the charge of the formed
carboxylate. In all cases, the product formed in the hydrolysis was d4TMP and the
corresponding salicyl alcohols. No cleavages of the ester groups were observed.
In contrast, in CEM/0 extracts studies acetate ester was cleaved in the case of
triester 5 (t1/2 = 3.8 h vs. 13.6 h in PBS), 5 fast (2.6 h vs. 7.9 h), and 5 slow (5.2 h
vs. 18.1 h). As well as in the chemical hydrolysis, the 5 slow isomer was more
stable than 5 fast. In all cases, the product of the enzymatic hydrolysis, the alcohol
3-OHEt-cycloSal-d4TMP 1, was detected by HPLC-co-elution experiments. The
esters 2 which should release the acid 3-HPr-cycloSal-d4TMP 1 were found to be
resistant to enzymatic cleavage. This difference was unexpected because carboxyl
groups are often bioreversibly blocked by esterification.
In liver extracts the stability of the acetates 5 were 26- to 60-fold lower than
those obtained in PBS! Here also the alcohol 3-OHEt-cycloSal-d4TMP 4 was formed
in the hydrolysis. Obviously, the concentration of the involved enzymes is much
higher in the liver extracts as compared to the CEM extracts. In this case, no
difference between the half-lives of the diastereomers 5 fast and slow was de-
tectable. The methyl ester in triesters 2 was again not cleaved.
Finally, the ‘‘lock-in’’ modified cycloSal-d4TMP’s were tested for their anti-HIV
activity in wild-type CEM- and mutant thymidine-kinase deficient cells (CEM/TKÀ;
Table 1). The parent nucleoside d4T was found to be active against HIV-1 and
HIV-2 but lost its activity in the mutant CEM-cells. The same has been found for
the acid 3-HPr-cycloSal-d4TMP 5, which can be attributed to an incapability of
membrane penetration due to the charged carboxylate. The acetate esters 5
showed good activities against HIV-1 and HIV-2 in wild-type cells. The isomer
3-AcEt-cycloSal-d4TMP 5 slow was 2- to 3.5-fold more activity than the isomer 5
fast. In the mutant cell line (CEM/TKÀ) full retention of antiviral activity was
observed (TK-bypass). For the methyl ester triesters 2 the same was found, except
that 2 fast lost some of its activity in the mutant CEM-cells.
REFERENCES
1. Meier, C. CycloSal-pronucleotides—design of chemical trojan horses. Mini Reviews Med. Chem. 2002, 2,
219–234.
2. Meier, C.; Ruppel, M.F.; Vukadinovi´c, C.; Balzarini, J. Second generation of the cycloSal-pronucleotides with
esterase-cleavable site: the ‘‘lock-in’’ concept. Nucleosides Nucleotides 2004, 23, 89–115.
3. Meier, C.; Lorey, M.; De Clercq, E.; Balzarini, J. CycloSal-2’,3’-dideoxy-2’,3’-didehydrothymidine mono-
phosphate (cycloSal-d4TMP): synthesis and antiviral evaluation of a new d4TMP delivery system. J. Med.
Chem. 1998, 41, 1417–1427.
4. Breton, G.W. Selective monoacetylation of unsymmetrical diols catalysed by silica gel-supported sodium
hydrogen sulfate. J. Org. Chem. 1997, 62, 8952–8954.
5. Brechbu¨hler, H.; Bu¨chi; Hatz, E.; Schreiber, J.; Eschenmoser, A. Die Reaktion von Carbons¨auren mit Acetalen
des N,N-Dimethylformamids: Eine Veresterungsmethode. Helv. Chim. Acta 1965, 48, 1746–1771.