New developments of the "lock-in" modified cycloSal-d4TMPs

Article abstract of DOI:10.1080/15257770701530707

New developments of the "lock-in" modified cycloSal-d4TMP are reported. Novel prodrugs with variations in the linker chain were introduced. The synthesis, hydrolysis properties in different media (PBS, CEM/0- and liver extracts) and the antiviral activities against HIV are shown. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701530707

Nucleosides, Nucleotides, and Nucleic Acids, 26:1325–1328, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701530707
NEW DEVELOPMENTS OF THE “LOCK-IN” MODIFIED
cycloSal-d4TMPs
Dalibor Vukadinovic´-Tenter
of Science, University of Hamburg, Hamburg, Germany
Department of Chemistry, Organic Chemistry, Faculty
2
Jan Balzarini
Chris Meier
Rega-Institute for Medical Research, K. U. Leuven, Leuven, Belgium
2
Department of Chemistry, Organic Chemistry, Faculty of Science,
2
University of Hamburg, Hamburg, Germany
New developments of the “lock-in” modified cycloSal-d4TMP are reported. Novel prodrugs with
2
variations in the linker chain were introduced. The synthesis, hydrolysis properties in different media
(PBS, CEM/0- and liver extracts) and the antiviral activities against HIV are shown.
Keywords CycloSal-pronucleotides; prodrugs; enzymatic cleavage
INTRODUCTION
The cycloSal-pronucleotides are well-established prodrugs for a deliv-
ery of bioactive nucleotides.[1] For these lipophilic prodrugs a passive
membrane transport into cells is supposed. So, it can not be excluded
that an unfavorable equilibrium is formed through the membrane. There-
fore, modified cycloSal-triesters were designed to trap the compounds
inside cells. To achieve this goal, triesters bearing esterase-cleavable
sites attached via an alkyl-linker to the cycloSal-moiety were synthesized.
After metabolism a polar cycloSal-NMP, e.g., carboxylate or alcohol,
will be released. This should prevent an efflux from the cells and ac-
cumulate the compounds inside (“lock-in”; Figure 1).[2] Our previous
work dealt with various alkyl ester and a C2-alkyl-linker attached either
in 3- or 5-position to the masking unit. The prodrugs, leading to an
alcohol after enzymatic cleavage, were good substrates for esterases.
Unfortunately, the formed alcohols were not polar enough for an ef-
ficient “lock-in.” Esters releasing a carboxylate showed no enzymatic
Address correspondence to Chris Meier, Department of Chemistry, Organic Chemistry, Faculty
of Science, University of Hamburg, Martin Luther King-Platz 6, 20146 Hamburg, Germany. E-mail:
laborhenry@gmx.de
1325

1326
D. Vukadinovic´-Tenter et al.
FIGURE 1 Proposed “lock-in” mechanism and “lock-in” modified cycloSal-d4TMPs.
cleavage.[3] So, the idea was to lengthen the linker chain to improve
substrate properties of the ester-modified prodrugs. Spacers with C3- and
C4-chains bearing methyl- and benzyl esters were introduced.
RESULTS
Starting material for the synthesis of the new “lock-in”-modified cycloSal-
d4TMPs were 3-butenoic and 4-pentenoic acid and 5-bromosalicyl alde-
hyde. The acids were converted via DCC-activation into the corresponding
methyl- and benzyl ester. The tert-butyl ester was prepared by reaction of
lithium tert-butylate and 4-pentenoic acid chloride. 5-bromosalicyl aldehyde
was reduced with LiAlH4 to yield 5-bromosalicyl alcohol. To avoid side re-
actions the alcohol functions were protected as an isopropyliden acetale
by transacetalisation with 2,2-dimethoxypropane. Hydroboration of the
3-butenoates and 4-pentenoates with 9-BBN gave the corresponding tri-
alkylboranes. These compounds were used without further purification
(one pot) in a Suzuki cross coupling reaction with 5-bromosalicyl al-
cohol isopropyliden acteale and Pd(dppf) as catalyst. The obtained es-
ter modified salicyl alcohol isopropyliden acetals were deproctected by a
catalytic amount of hydrochloric acid to yield the desired salicyl acohol
derivates.[4] These diols were reacted with PCl3 to yield cyclic chlorophos-
phanes, which were then used as phosphitylation agents of d4T. The result-
ing phosphites were oxidized to give cycloSal-triesters 1-3. Finally, deprotec-
tion of 3-t-BuPe-cycloSal-d4TMP with TFA led to 3-COOHBu-cycloSal-d4TMP
3a.
All cycloSal-prodrugs 1-3 were analyzed according to their hydrolysis half-
lives in different media (PBS, CEM/0- and liver extracts) and their anti-HIV
activities. In chemical hydrolysis (PBS) half-lives t1/2 of 4-6 h for the ester

“Lock-In” Modified cycloSal-d4TMPs
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TABLE 1 Hydrolysis half-lives and anti-HIV activities
t1/2 [h]
EC50 [µM]
pH
7.3 CEM/O Liver
CEM/0
HIV-1
CEM/0
HIV-2
CEM/TK− CC50 [µM]
Compound
n
R
HIV-2
CEM/0
1a
1b
1c
2
3a
3b
d4T
1
1
1
2
3
3
-COOH 12.5
-COOMe 5.7
11.4
7.0
6.5
5.7
6.8
6.0
n.a.
n.d. 0.14 0.10 0.80 0.20 50.0 30.0
n.d. 0.33 0.11 1.05 0.35 1.20 1.10 58.1 1.84
n.d. 0.08 0.00 1.00 0.28 2.00 0.00 60.6 3.30
76
-COOBn
4.3
-COOMe 4.3
3.8 0.26 0.14 0.20 0.10
9.8 n.d. n.d.
0.1 0.37 0.20 0.31 0.08
3.5
n.d.
>10
74.4 5.30
n.d.
68.1 11.7
-COOH
-COOBn
9.0
4.0
n.a.
n.a. 0.23 0.04 0.24 0.02 15.0 7.1 58.8 24.2
n.d.: not determined; n.a.: not available.
1b-c, 2, and 3b were determined. These values have been expected, because
the negative inductive effect of the ester groups is separated by the alkyl-
spacer from the aromatic moiety. The increased stabilities for the acids 1a
(t1/2 = 12.5 hours) and 3a (t1/2 = 9.0 hours) may be attributed to the charge
of the carboxylates at pH 7.3. An overall negative charge of the molecule
may interfere with the nucleophilic attack at the phosphate group. Hydrol-
ysis studies in human CEM/0 extracts revealed that all esters 1-3 showed
no enzymatic cleavage in this medium. The product of the hydrolysis was
d4TMP, not the corresponding acids 1a and 3a. Due to the lower pH value
(6.8) the half-lives for 1-3 were higher than those found in PBS. In con-
trast in mouse liver extracts an enzymatic reaction for 3b was observed.
The hydrolysis half-life (t1/2 = 0.1 hour) was 40-fold lower than in chemi-
cal media (PBS t1/2 = 4.0 hours). HPLC-co-elution experiments showed un-
ambiguously that the carboxylate 3a was the main hydrolysis product. The
compounds 1b-c and 2 were not tested or showed no enzymatic reaction.
Finally, the “lock-in”-cycloSal-d4TMPs were tested for their antiviral activities
against HIV-1 and HIV-2 in wild-type CEM- and in mutant thymindine kinase
deficient cells (CEM/TK−, Table 1). The parent nucleoside d4T was found
to be active against HIV-1 and HIV-2 but it lost its activity in the mutant
CEM cells. The same has been found for acid 1a, which can be explained by
an incapability of membrane penetration due to the charged carboxylate.
The found activity of 1a wild-type cells (CEM/0) derives from the parent
nucleoside d4T, which is generated by chemical hydrolysis of the prodrug
and a subsequent dephosphorylation of d4TMP outside cells. The esters 1b,
2, and 3b showed good activities against HIV-1 and HIV-2 (0.2–1.0 µM) in
wild-type cells. The benzyl-ester 1c was very potent (0.08 µM) against HIV-1
and 10-fold lower potency against HIV-2 (1.0 µM). Surprisingly, only for
1b and 1c full retention of the activity in the mutant cell-line was observed
(TK-Bypass). The triester with C3- (2) or C4-alkyl-linker (3b) lost their an-
tiviral activities in TK-deficient cells.

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CONCLUSION
D. Vukadinovic´-Tenter et al.
A new synthesis of “lock-in” modified cycloSal-d4TMPs has been estab-
lished and applied in order to vary the linker length of the prodrugs. The
introduction of C3- and C4-alkyl-linker to the cycloSal-moiety did not lead to
a significant improvement of esterase affinity.
REFERENCES
1. Meier, C. cycloSal-pronucleotides—Design of chemical trojan horses. Mini Rev. Med. Chem. 2002, 2,
219–234.
2. Meier, C.; Ruppel, M.F.H.; Vukadinovic´, D.; Balzarini, J. “Lock-in”-cycloSal-pronucleotides—A new
generation of chemical Trojan Horses? Mini Rev. Med. Chem. 2004, 4(4), 383–394.
3. 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(1–2),
89–115.
4. Meier, C.; Ducho, C.; Jessen, H.; Vukadinovic´-Tenter, D.; Balzarini, J. Second generation of cycloSal-
d4TMP pronucelotides bearing esterase cleavable sites—The trapping concept. Eur. J. Org. Chem.
2006, 1, 197–206.