5-Diacetoxymethyl-cycloSal-d4TMP - A prototype of enzymatically activated cyclosal-pronucleotides

Article abstract of DOI:10.1080/15257770701504025

A new class of "lock-in"-modified cycloSal-pronucleotides has been synthesized. On the example of 5-diacetoxymethyl-cycloSal-d4T-monophosphate (5-di-AM-cycloSal-d4TMP), the concept of enzymatically activated cycloSal-pronucleotides is elucidated. Synthesis, hydrolysis studies, and antiviral activities against HIV are presented. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701504025

Nucleosides, Nucleotides, and Nucleic Acids, 26:861–864, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701504025
5
-DIACETOXYMETHYL-CYCLOSAL-D4TMP—A PROTOTYPE OF
ENZYMATICALLY ACTIVATED CYCLOSAL-PRONUCLEOTIDES
N. Gisch
2
Department of Chemistry, Organic Chemistry, Faculty of Science, University
of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
J. Balzarini Rega-Institute for Medical Research, K.U. Leuven, Minderbroedersstraat
2
1
0, 3000 Leuven, Belgium
C. Meier
2
Department of Chemistry, Organic Chemistry, Faculty of Science, University
of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
2
A new class of “lock-in”-modified cycloSal-pronucleotides has been synthesized. On the exam-
ple of 5-diacetoxymethyl-cycloSal-d4T-monophosphate (5-di-AM-cycloSal-d4TMP), the concept of
enzymatically activated cycloSal-pronucleotides is elucidated. Synthesis, hydrolysis studies, and an-
tiviral activities against HIV are presented.
Keywords Pronucleotides; cycloSal; anti-HIV; enzymatic activation; antiviral activity
INTRODUCTION
One established concept for the delivery of therapeutically active nucle-
ꢁ
ꢁ
ꢁ
ꢁ
oside monophosphates (NMPs) like 2 ,3 -dideoxy-2 ,3 -didehydrothymidine
[1]
monophosphate (d4TMP, 1) into cells is the cycloSal-concept. This pro-
drug concept has been successfully applied to a multitude of nucleoside
analogs. Due to the chemically triggered delivery mechanism[3] and the
lipophilic character of cycloSal-triester a concentration equilibrium formed
through the cell membrane is supposed. This is unfavorable for antiviral
efficiency because a high intracellular concentration of the pronucleotide
is necessary. Therefore “lock-in”-modified cycloSal-pronucleotides have been
developed. These pronucleotides bear a (carboxy)esterase-cleavable ester
site attached to the aromatic ring in order to trap the cycloSal-triester in-
side cells by cleavage of the ester group to release a highly polar derivate.
To avoid a considerable reduction of the chemical stability of the cycloSal-
triester, these groups have been separated from the aromatic ring by an
[2]
Address correspondence to N. Gisch, University of Hamburg, Martin-Luther-King-Platz 6, Hamburg
20146, Germany. E-mail: gisch@chemie.uni-hamburg.de
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N. Gisch et al.
FIGURE 1 Concept of enzymatically activated cycloSal-Pronucleotides shown on the example of 5-
diacetoxymethyl-cycloSal-d4TMP 2.
alkyl spacer. It has been shown that an effective intracellular trapping
should be possible, if highly polar cycloSal-d4TMP acids are released from
cycloSal-d4TMP acid ester. From 5-propionyl-cycloSal-d4TMP acid, d4TMP
was released by the known chemically induced pathway. However, these
“
lock-in”-compounds led to a delayed drug delivery due to high chemical
[
4,5]
stability.
Here, we present a prototype of the new concept of enzymat-
ically activated cycloSal-pronucleotides, 5-diacetoxymethyl-cycloSal-d4TMP 2.
In this concept, after passive transport of 2 into cells (step c, Figure 1), the
lipophilic acylal substituent having a weak electron-withdrawing effect at-
tached to the aromatic ring is converted into a highly polar aldehyde group
with a strong electron-withdrawing effect by intracellular cleavage (step d,
Figure 1).
The formed acceptor group leads to a strong decrease in hydrol-
ysis stability and the rapid formation of a charged intermediate 4
phosphodiester),[ so that compound 3 should not be effluxed (step e, Fig-
3]
(
ure 1) and the phosphodiester intermediate 4 should not be effluxed at all
due to the resulting polarity. From the phosphodiester d4TMP 1 is released
subsequently (step f, Figure 1). This concept is based on the higher intra-
[6]
cellular concentration of esterases compared to the extracellular medium
Figure 1, step b should not take place) and on a considerable difference
(

Prototype of Enzymatically Activated cycloSal-Pronucleotides
863
SCHEME 1 Synthesis of 5-diacetoxymethyl-cycloSal-d4TMP 2. Method A: THF, LiAlH4, room temp. to
◦
reflux, 3 h, 91%; method B: acetone, 2,2-dimethoxypropane, p TsOH, Na2SO4, 40 C, 3 d, 95%; method
◦
C: THF, n-BuLi, DMF, –78 C, 3 h, 94%; method D: CH3CN/H2O, cat. HCl, 81%; method E: i) THF, PCl3,
pyridine,–20 C to room temp., 4.5 h; ii) CH3CN, DIPEA, d4T, –20 C to room temp., 3 h; iii) CH3CN,
tBuOOH, –20 C to room temp., 1 h, 31%; method F: CH3CN, acetic anhydride, ZrCl4; room temp., 45
◦
◦
◦
min, 44%.
between the extracellular hydrolysis stability of the 5-diacetoxymethyl-
cycloSal-d4TMP 2 and the intracellular hydrolysis stability of the
5-formyl-cycloSal-d4TMP 3 after enzymatical cleavage (Figure 1, t1/2 step a
ꢂ t1/2 step d).
RESULTS
The title compound 2 was synthesized starting from the commercial
available 5-bromosalicyl aldehyde 5 as outlined in Scheme 1.
Reduction of 5 gave the 4-bromosalicyl alcohol 6 in 91% yield. After
protection of 6 as isopropylidene acetal (7, 95% yield) the formyl group was
introduced via bromo-lithium exchange to yield 4-formylsalicyl alcohol iso-
propylidene acetal 8 in 94%. By means of acidic deprotection 4-formylsalicyl
alcohol 9 could be obtained in 81% yield. Compound 9 was converted to
3
(mixture of two diastereomeres) using chlorophosphite chemistry as de-
[1]
scribed before. The only modification was an exchange of the solvent.
The title compound 2 was synthesized by protection of the formyl group of
3
as an acylal in 44% yield.
The cycloSal-triesters 2 and 3 were studied for their stability in aqueous
5 mM phosphate buffer (pH = 7.3). As expected, 5-formyl-cycloSal-d4TMP
has a very short half-life of 0.18 h. The half-life of 5-diacetoxymethyl-
2
3
cycloSal-d4TMP 2 is 6-fold higher (t1/2 = 1.2 h). In the study of 2 no com-
peting hydrolysis of the acylal ester group was observed. So, the half-life
corresponds to the cleavage of the triesters and the exclusive formation of
31
d4TMP 1 (proven by P-NMR hydrolysis). The cleavage of the acylal group
of 2 was shown in hydrolysis studies in T-lymphocyte cell extracts. The half-
life was 0.08 h and the corresponding 5-formyl-cycloSal-d4TMP 3 was formed
(HPLC co-elution experiments). CycloSal-triester 2 and 3 were tested for

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N. Gisch et al.
their anti-HIV activity in wild type CEM/0 and mutant thymidine-kinase-
−
deficient CEM/TK cells. As reference d4T (active against HIV-1 and HIV-2
−
in CEM/0 but weakly active in CEM/TK cells) was used. Compounds 2 and
3
have the same activity against HIV-1 in wild-type cells and against HIV-2 in
CEM/0 cells as d4T (2: 0.42± 0.28 µM and 0.40± 0.0 µM; 3: 0.41± 0.29 µM
and 0.15± 0.08 µM; d4T: 0.48± 0.45 µM and 0.63± 0.21 µM).
−
The activity of triesters 2 and 3 against HIV-2 in CEM/TK cells is
2
5
4
6-fold, respectively, 33-fold lower as in CEM/0 cells (2: 10.5± 8.3 µM; 3:
.0± 4.6 µM). However, the activity of d4T decreases even 100-fold (d4T:
7.5± 26.3 µM). So, a partial delivery of the pronucleotide takes place. The
−
loss of antiviral activity in CEM/TK is comparable with the values for other
acceptor substituted cycloSal-d4TMPs with low hydrolysis half-lives.
[2]
In conclusion, a fast release of NMPs by enzymatic activation out of
cycloSal-prodrugs like compound 2 seems to be possible. Further work will
be done to increase the hydrolysis stability in order to achieve full retention
of antiviral activity.
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1.
2.
3.
4.
Meier, C. cycloSal-pronucleotides–Design of chemical trojan horses. Mini Rev. Medicinal Chem. 2002,
, 219–234.
2
Meier, C. cycloSal phosphates as chemical trojan horses for intracellular nucleotide and glycosyl-
monophosphate delivery–Chemistry meets biology. Eur. J. Organic Chem. 2006, 1081–1102.
Meier, C. 2004. cycloSal-Pronucleotides–Design of the concept, chemistry, and antiviral activity. In
Advances in Antiviral Drug Design, Vol. 4, E. de Clercq, Ed. Elsevier, Amsterdam, pp. 147–213.
Meier, C.; Ruppel, M.F.H.; Vukadinovic, D.; Balzarini, J. Second generation of cycloSal-pronucleotides
with esterase-cleavable sites: The “lock-in”-concept. Nucleosides, Nucleotides & Nucleic Acids 2004, 23,
89–115.
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.
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Meier, C.; Ducho, C.; Jessen, H.; Vukadinovi-Tenter, D.; Balzarini, J. Second-generation cycloSal-
d4TMP pronucleotides bearing esterase-cleavable sites–The “trapping” concept. Eur. J. Organic Chem.
2006, 197–206.
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Perigaud, C.; Gosselin, G.; Imbach, J.-L. Minireview: from the pronucleotide concept to the SATE
phosphate protecting groups. Current Topics Med. Chem. 1997, 2, 15–29.