Figure 1: General metabolism of nucleoside analogues and kinase bypass of nucleoside monophosphates.
this context we have developed the so-called cyclo
Sal-pronucleotide approach that is one of the leading
pronucleotide systems world-wide.
The basic idea is a highly
selective chemically driven hydrolysis mechanism that delivers nucleotides into
cells. As lipophilic masking unit a salicylalcohol is covalently attached to
the two remaining acid functionalities of the nucleotide. By intelligent
construction of this system, the first hydrolysis step leaves a highly reactive
intermediate that spontaneously breaks down to yield the nucleotide (first
generation compounds). The delivery mechanism leads selectively to nucleotides
avoiding the known pseudorotational processes at the phosphorus atom. The
mechanism depicted in the following scheme has been proven by 31
studies, HPLC-based studies as well as isotope-labelling and was characterized
in kinetic studies as well.[2-4]
Figure 2: Hydrolysis mechanism of cyclo
approach has been applied successfully to various nucleoside analogues proving
that the antiviral activity can be improved, resistance towards a parent
nucleoside can be overcome and even inactive nucleoside analogues can be
converted into bioactive derivatives.[5-9]
to the lipophilic character of cyclo
triesters and their chemically-triggered delivery mechanism, a drug
concentration equilibrium is formed through the cell
membrane. In order to trap the lipophilic cyclo
inside cells, so called “lock-in”-cyclo
designed (second generation compounds).
These compounds are bearing esterase-cleavable esters attached to the aromatic
ring. To avoid any influence on the chemical hydrolysis properties, the ester
moieties are separated from the aromatic ring by a C2
Sal-d4TMP acid ester and cyclo
Sal-d4TMP alcohol ester
as well as acylal
or amino acid ester
Sal-d4TMPs have been investigated.
Hydrolysis studies in phosphate buffer (PBS, pH=7.3) and in T-lymphocyte CEM
cell extracts revealed that an intracellular trapping is possible, if highly
polar, charged cyclo
are released. The fast intracellular release of these compounds is the primary
aim of the “lock-in”-concept. D4TMP is released from the cyclo
Sal-d4TMP acids by chemical hydrolysis next. However, this
release was found to be very slow.
Figure 3: General structure of "lock-in"-cyclo
as a conceptually different extension, enzymatically
have been developed (third generation prodrugs). I
n this approach, prodrugs also have a lipophilic, functionalized
substituent with electron-donating or only weak electron-withdrawing properties
attached to the aromatic ring of the cycloSal-mask.
After passive transport into cells the functionalized substituent is converted
into a strong electron-withdrawing group by enzymatic cleavage. This group causes
a significant decrease in hydrolysis stability of the cycloSal-compound. In contrast to the “lock-in” approach, in this
case the enzymatic triggering results in a fast delivery of the nucleotide,
instead of a highly polar, chemically more stable intermediate. Thus, the trapping
idea with these compounds is realized on the nucleotide level.
Figure 4: General structure of enzymatically activated cyclo
As enzymatically activated cycloSal-prodrugs
as well as 5-(1-acetoxyvinyl)-cycloSal-d4TMPs
have been developed.
Recently, we have extended the cycloSal-pronucleotide approach to the first synthesis of so-called
“high-loaded” pronucleotides (fourth generation compounds).[15,16] In
the original concept the ratio of the masking unit to the nucleotide was
1:1. In order to increase this ratio so
that more nucleotides are attached to the vector, tetrols or dimers of
salicylalcohols were used and charged with two equivalents of the corresponding
nucleoside. It was shown that such compounds indeed deliver step-wise two
nucleotide molecules by chemical hydrolysis. These compounds also proved to be
active in a mutant T-lymphocyte cell line that is used for the anti-HIV tests.
Figure 5: General structure of "high-loaded"-cyclo
2. Prodrugs of Nucleoside diphosphates
Figure 6: General metabolism of nucleoside analogues and kinase bypass of nucleoside diphosphates.
contrast to prodrugs of nucleotides, the development of nucleoside diphosphate
or even triphosphate prodrugs has only been addressed in very few cases. This
is remarkable, because it is known that e.g. 3´-azido-3´-deoxythymidine (AZT),
the first approved nucleosidic anti-HIV drug, is only very slowly
diphosphorylated by thymidylate kinase.
Further, the resulting accumulation
of AZT monophosphate (AZTMP) provokes severe side-effects.
we have reported on the first successful nucleoside diphosphate prodrug
In contrast to the cyclo
here the delivery mechanism relies on an enzymatically triggered process.
Chemical methods did not lead to a selective
release of the corresponding nucleoside diphosphate. Bis-(acyloxybenzyl)esters
(BAB-esters) of the nucleoside diphosphate were prepared and showed very
promising and exciting results referring to the bioreversible deprotection of
this class of highly polar metabolites. The lead structure of these prodrugs is
shown in the scheme below as well as the proposed mechanism of hydrolysis.
Figure 7: Enzymatic cleavage of Bis-BAB nucleoside diphosphate prodrugs.
- C. Meier, CycloSal-Phosphates as Chemical Trojan
Horses for the Intracellular Nucleotide and Glycosylmonophosphate Delivery –
Chemistry meets Biology, Eur. J. Org.
Chem. 2006, 1081-1102.
- C. Meier, A.
Meerbach, J. Balzarini, cycloSal-Pronucleotides
– Development of First and Second Generation Chemical Trojan Horses for Antiviral Chemotherapy, Frontiers in
Bioscience 2004, 9, 873-890.
- C. Meier, M.
Ruppel, D. Vukadinovic, J. Balzarini, “Lock-in”-cycloSal-pronucleotides – A New Generation of Chemical Trojan Horses, Minirev.
Med. Chem. 2004, 4, 383-394.
- J. Balzarini, S.
Aquaro, T. Knispel, C. Rampazzo, V. Bianchi, C.-F. Perno, E. De Clercq, C.
Efficient Intracellular Delivery of d4TMP,
Mol. Pharmacol. 2000, 58, 928-935.
- C. Meier, A. Lomp,
A. Meerbach, P. Wutzler, CycloSal-BVDUMP-Pronucleotides – How to Convert an anti-EBV-inactive Nucleoside Analogue into a Bioactive Compound, J. Med. Chem. 2002,
- C. Meier, T.
Knispel, E. De Clercq, J. Balzarini, CycloSal-Pro-Nucleotides
2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d4A):
Synthesis and Antiviral Evaluation of a Highly Efficient Nucleotide Delivery System, J. Med. Chem. 1999, 42, 1604-1614.
- C. Meier, T.
Knispel, V. E. Marquez, M. A. Siddiqui, E.
De Clercq, J. Balzarini, CycloSal-Pro-Nucleotides
of 2'-fluoro-ara- and 2'-fluoro-ribo-2',3'-dideoxyadenosine (F-ara- and
F-ribo-ddA) as a Strategy to Bypass a Metabolic Blockade, J. Med. Chem. 1999, 42, 1615-1624.
- A. Sauerbrei, C.
Meier, A. Meerbach, P. Wutzler, Inhibition Efficiency of cycloSal-Nucleoside Monophophates of Aciclovir and Brivudine on DNA
Synthesis of Orthopoxviruses, Antiviral
Chem. Chemother. 2006, 17, 25-31.
- C. Meier, U. Görbig,
C. Müller, J. Balzarini, CycloSal-PMEA and cycloAmb-PMEA
– Potentially new Phosphonate Prodrugs on the Basis of the cycloSal-Pronucleotide Approach, J. Med. Chem. 2005, 48, 8079-8086.
- C. Meier, M. F. H.
Ruppel, D. Vukadinović, J. Balzarini, Second Generation of cycloSal-Pronucleotides with Esterase-Cleavable Sites: The
Nucleotides Nucleic Acids 2004,
- C. Meier, C. Ducho,
H. Jessen, D. Vukadinović-Tenter, J. Balzarini, Second-Generation cycloSal-d4TMP Pronucleotides Bearing
Esterase-Cleavable Sites – The „Trapping“ Concept, Eur. J. Org. Chem. 2006,
- H. J. Jessen, J.
Balzarini, C. Meier, Intracellular Trapping of cycloSal-Pronucleotides: Modification of Prodrugs with Amino Acid
Esters, J. Med. Chem. 2008, 51, 6592-6598.
- N. Gisch, J.
Balzarini, C. Meier, Studies on Enzyme-cleavable Dialkoxymethyl-cycloSaligenyl-2’,3’-dideoxy-2’,3’-didehydrothymidine
Monophosphates, J. Med. Chem. 2008, 51, 6752-6760.
- N. Gisch, F.
Pertenbreiter, J. Balzarini, C. Meier, 5-(1-Acetoxyvinyl)-cycloSaligenyl-2’,3’-dideoxy-2’,3’-didehydrothymidine
Monophosphates – A Second Type of New, Enzymatically Activated cycloSal-Pronucleotides, J. Med. Chem. 2008,
- C. Ducho, U.
Görbig, S. Jessel, N. Gisch, J. Balzarini, C. Meier, Bis-cycloSal-d4T-monophosphates
– Drugs that Deliver Two Molecules of Bioactive Nucleotides, J. Med. Chem. 2007, 50, 1335-1346.
- N. Gisch, J.
Balzarini, C. Meier, Doubly loaded cycloSaligenyl-Pronucleotides
monophosphates), J. Med. Chem. 2009, 52, 3464-3473.
- H. J. Jessen, T.
Schulz, J. Balzarini, C. Meier, Bioreversible protection of
nucleosidediphosphates, Angew. Chem. Int.
Ed. Engl. 2008, 47, 8719-8722.