"Cascade-release dendrimers" liberate all end groups upon a single triggering event in the dendritic core

Article abstract of DOI:10.1002/anie.200351942

A single triggering event in the core of a dendrimer composed of two or more generations of multiple-release monomeric building blocks can induce the release of all end groups at the termini of the dendrimer (see schematic representation). Such multiple-release systems could find application, for example, in the field of (targeted) drug delivery, where a disease- or organ-specific activation event could trigger simultaneous release of multiple biologically active molecules.

Full text of DOI:10.1002/anie.200351942

Communications
Cascade-Release Dendrimers
reactions, thereby releasing all the end groups from the
periphery of the “exploding” cascade-release dendrimer (see
Supporting Information).
“Cascade-Release Dendrimers” Liberate All End
Groups upon a Single Triggering Event in the
Dendritic Core**
Functional dendrimers of this kind may find application in
several fields. One particular area is (targeted) drug delivery,
where cascade-release dendrimers possess two major advan-
tages over conventional dendrimers: 1) multiple covalently
bound drug molecules can be site-specifically released from
the targeting moiety by a single cleaving step; and 2) they are
selectively as well as completely degraded. Thus, in contrast to
conventional dendrimers, they can be easily drained from the
body. Clearance of macromolecules from the body may be a
limiting factor in current macromolecular drug-delivery
systems,[15] as the long-term effects of some synthetic macro-
molecules in parenteral medicine are not completely
known.[16]
To facilitate drug release, linkers are frequently incorpo-
rated between the drug molecule and a cleavable carrier,
which is termed the “specifier” (Figure 1).
Cascade-release dendrimers are constructed from two or
more generations of branched self-elimination linkers, each of
which releases multiple leaving groups after a single activa-
tion (Figure 2). We present here two such structurally distinct
monomeric multiple-release building blocks. Both linkers are
based on a general type of single-release linker that is
eliminated as a consequence of shifting conjugated electron
Franciscus M. H. de Groot,* Carsten Albrecht,
Ralph Koekkoek, Patrick H. Beusker, and
Hans W. Scheeren
Dendrimers are well-defined, branched treelike molecules
with multiple end groups.[1,2] Numerous applications of these
starlike structures have been explored.[3,4] They can be
utilized in areas such as magnetic resonance imaging (MRI),
gene therapy,[5] liquid crystals, sensors, and catalysis. The
application of dendrimers in drug delivery is currently
receiving much scientific attention.[6–9] Most dendrimers
reported so far have been constructed as a branched skeleton
that incorporates functional molecules either covalently or
noncovalently. Biologically active substances that are
attached to the termini of dendritic structures[10] can be
liberated by chemical or biological methods.[11] Several
branched constructs have been reported which contain
covalently linked doxorubicin molecules as end groups.[12–14]
However, in all the branched structures or dendrimers
reported so far, each single
drug has to be independently
cleaved to be released. Here,
we report dendrimers that
have been built to completely
and rapidly dissociate into
separate building blocks
Figure 1. A conjugated drug (gray) is released from an inactive conjugate. Cleavage between the specifier
(green) and the linker (blue) induces spontaneous self-elimination of the linker, thus releasing the drug
molecule (red) with regained activity.
upon
a
single triggering
event in the dendritic core.
This process induces simulta-
neous release of all end-group
molecules connected to the dendritic termini. We term these
multiple-release dendritic systems “cascade-release dendrim-
ers”. Such dendrimers collapse into their separate monomeric
building blocks after a single (chemical or biological)
activation step that triggers a cascade of self-elimination
[*] Dr. F. M. H. de Groot, Dr. P. H. Beusker
Syntarga B.V.
Toernooiveld, 6525 ED Nijmegen (The Netherlands)
Fax: (+31)24-365-2929
E-mail: fmhdegroot@syntarga.com
C. Albrecht, R. Koekkoek, Dr. H. W. Scheeren
Department of Organic Chemistry
University of Nijmegen
Toernooiveld 1, 6525 ED Nijmegen (The Netherlands)
[**] We thank NWO-ZonMw for a STIGON grant. Prof. J. C. M. van Hest,
Dr. F. L. van Delft, and Prof. R. J. M. Nolte are thanked for their
helpful comments. Dr. D. de Vos (Pharmachemie B.V., Haarlem,
The Netherlands) is acknowledged for a gift of paclitaxel. We are
grateful to Dr. H. Engelkamp for assistance in preparing Figures 1
and 2.
Figure 2. A single activation of a second generation cascade-release
dendrimer triggers a cascade of self-eliminations and induces release
of all end groups. Covalently bound end groups are depicted in gray,
branched self-elimination linkers in blue, and the specifier in green.
The released end groups are depicted in red.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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DOI: 10.1002/anie.200351942
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pairs, which leads to expulsion of the
leaving group (for example, a drug). The
most prominent example of this self-elim-
ination linker type is the highly versatile
and well-known 4-aminobenzyl alcohol
linker.[17] We recently reported that the
elongation of this conventional 1,6-elimi-
nation linker results in enhanced rates of
drug release.[18] It was shown that the
elongated 4-aminocinnamyl alcohol linker
released paclitaxel (taxol) following 1,8-
elimination.[19] Herein, proof of principle
is reported for both the release of two
leaving groups from a branched double-
release linker, which can be obtained by
introduction of branching into the 4-ami-
nocinnamyl alcohol linker, and for the
release of three leaving groups from a
branched triple-release linker, which is
based on the 1,6-elimination linker. Fur-
thermore, we demonstrate that a cascade-
release dendrimer which contains two
generations of double-release linkers com-
pletely falls apart into its free monomers
and releases all end groups upon a single
activating event.
Scheme 1. Structure of double-release linker 1 and the proposed mechanism of elimination of both
leaving groups L upon activation of 2.
The double-release self-elimination
AB2-type monomer 2-(4-aminobenzylide-
ne)propane-1,3-diol (1) and the proposed
mechanism of the release of two leaving
groups upon activation are depicted in
Scheme 1. The multiple-release system is
stable as long as the amine function in 2 is
capped by a protecting group. Unmasking
(step a) the protected amine 2 triggers two
1,8-elimination reactions from amine 3, in
which two molecules of CO2 and two
leaving groups L are liberated. The inter-
mediate non-aromatic species 4 that is
formed after the first self-elimination
(step b) is trapped by a nucleophile, such
as water, in step c to regenerate an aro-
matic species 5 that can undergo the
second self-elimination (step d). Quench-
ing of the intermediate with water (for
example, under physiological conditions)
will generate aminodiol building block 1.
Proof of concept for complete release
of both end groups from the double-
release linker was firmly established in a
branched system in which nitrodiol 6[20]
formed the core (Scheme 2). The nitro
function can be considered as a masked
amine. Chemical reduction of the nitro
Scheme 2. Synthesis of branched construct 8 and elimination of two paclitaxel (taxol) molecules upon
a single triggering reaction (reduction of the nitro function). DIPEA=N,N,-diisopropylethylamine,
DMAP=4-dimethylaminopyridine, Bz=benzoyl.
group to the amine should trigger the cascade of self-
eliminations. Nitrodiol 6 was activated using 4-nitrophenyl
chloroformate to give the corresponding bis(4-nitrophenyl
carbonate) 7, which was subsequently coupled with two
equivalents of paclitaxel to yield dendron 8. To demonstrate
the generality of the concept, the chemotherapeutic drug
paclitaxel was chosen as a model compound because of its
complex structure and bulkiness. At 08C paclitaxel reacted
specifically with the activated carbonates through its most
reactive 2’-hydroxy function. The nitro function was then
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reduced under mild conditions (Zn, acetic acid; Scheme 2).
Mass spectrometric studies showed that compound 8, its
reduced congener, or any of the intermediates similar to the
ones in Scheme 1 were no longer present, and clearly showed
a major peak corresponding to free paclitaxel. No degrada-
tion or formation of side products had taken place (TLC,
NMR), thus showing the validity of the concept. The kinetics
for electronic cascade self-eliminations are generally fast.[17]
Paclitaxel was immediately formed, which left the triggering
reaction as the rate-limiting step for complete dissociation.
With the double-release linker in hand, we now focused
on the construction of a dendritic structure to unequivocally
establish that multiple release occurred from a cascade-
release dendrimer. A divergent route, starting with the
dendrimer core (Scheme 3), was used to synthesize cascade-
Analysis by thin-layer chromatography indicated complete
disappearance of the starting material and formation of
unconjugated paclitaxel within 30 minutes. 1H NMR spectro-
scopic studies unambiguously confirmed the complete release
of both paclitaxel molecules (see Supporting Information).
Although the NMR spectra of large paclitaxel derivatives are
generally complicated, the 2’-H signal of 8 can be identified as
a clearly distinguishable signal at d = 5.46 ppm, which is
indicative of a 2’-hydroxy group functionalized with an
alkyloxycarbonyl group. This signal completely vanished
following the multiple-release cascade, thus indicating that
no conjugated paclitaxel was present, whereas the character-
istic paclitaxel 2’-H signal at d = 4.74 ppm was clearly present.
Scheme 3. Synthesis of second-generation cascade-release dendrimer 11 and release of four paclitaxel leaving groups upon reduction. HOBt=1-
hydroxy-1H-benzotriazole.
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Chemie
release dendrimer 11. Two generations of double-release
linkers were connected through carbamate linkages by
coupling two molecules of aminodiol 1 to the bis(4-nitro-
phenyl carbonate) 7 to yield 9. The four terminal alcohol
groups were then activated to give tetracarbonate 10, which
was coupled to four equivalents of paclitaxel to yield the
second-generation cascade-release dendrimer 11 loaded with
four paclitaxel molecules at the dendritic termini. To prove
the validity of the dendritic cascade-release concept, second
generation dendrimer 11 was subjected to reduction using Zn
and acetic acid (Scheme 3). Chromatographic analysis once
again indicated rapid and complete disappearance of the
starting compound and formation of paclitaxel. 1H NMR
spectroscopic analysis demonstrated complete formation of
liberated paclitaxel and no degradation products. No con-
jugated paclitaxel was present, thus demonstrating that 11
behaves as a cascade-release dendrimer that releases all end
groups according to the sequence depicted in Figure 2.
upon dendritic disintegration, for their cytotoxicity in a panel
of seven well-characterized human tumor cell lines. Neither
compound displayed any toxicity. Details concerning the
experimental procedures and in vitro cytotoxicity are
enclosed as Supporting Information.
Cascade-release dendrimers, as well as the multiple-
release monomers, may show utility in fields such as
(targeted) drug delivery, biodegradable materials, controlled
release, and diagnostics. Much room is left for the variation of
individual components for the design of a cascade-release
dendrimer with desired properties. The characteristics of the
activation can be modified by the choice of specifier. An
appropriate specifier can, for example, be a specific peptide
substrate for a disease-associated enzyme, or it may contain
an additional targeting moiety, such as, for example, an
antibody. Two or more different parent compounds can be
attached as end groups. Linear self-elimination linkers can be
incorporated between the specifier and the first branched
linker to improve the efficiency of drug release.[18] Linear self-
elimination linkers can be incorporated between generations
of branched monomers to increase the outer sphere surface
and the number of end groups that can be accommodated.
Cascade-release dendrimers can be used for drug delivery,
particularly for tumor-targeted drug delivery,[22,23] where they
may potentially induce a substantial therapeutic advantage
over single-release unbranched conjugates.[24–26] The cascade-
release dendrimer amplifies the effect of one tumor-specific
activating reaction, by triggering a “cytotoxic explosion”.
Cascade-release dendrimers of sufficient size will also pas-
sively target by the enhanced permeability and retention
(EPR) effect,[27] and, as for conventional dendrimers, they can
be manufactured as monodisperse and homogeneous com-
pounds. The monomers presented here are appropriate for
tumor targeting, because self-elimination of the double- and
triple-release monomers proceeds with a short half-life,[17,18]
and because the monomers can be connected through
carbamate linkages that are generally stable under physio-
logical conditions.[28] We have reported chemical proof of the
concept of cascade-release dendrimers. Their potential in
tumor targeting should be exploited, for example, by choosing
a specifier that is a substrate for tumor-associated or tumor-
targeted enzymes and a highly toxic anticancer agent for
attachment at the dendritic termini.
We have also designed and synthesized a second multiple-
release linker, (2-amino-3,5-di(hydroxymethyl)phenyl)me-
thanol (12, Scheme 4). This triple-release linker is an aniline
Scheme 4. Structures of triple-release linker 12 and its functionalized
nitroaromatic derivative 13.
derivative with a hydroxymethyl group at the 2-, 4-, and 6-
positions, and it combines one 1,6- with two 1,4-elimination
systems in a single linker.[17] We synthesized a nitrotriol
derivative of this AB3-type monomeric building block[21] (13)
containing three phenethyl alcohol molecules as leaving
groups connected by carbonate linkages. All the starting
material was, again, completely converted into free phenethyl
alcohol (MS, NMR) upon reduction of the nitro function, thus
validating aminotriol 12 as a triple-release monomeric linker.
To prove that the conditions used for the reduction of the
nitro function in the synthesized compounds did not directly
effect cleavage of the paclitaxel carbonate or phenethyl
carbonate linkages, we treated reference compounds dibenzyl
carbonate and 2’-O-(cinnamyloxycarbonyl)paclitaxel with
Zn/acetic acid. Both compounds remained fully intact under
these conditions (as observed by 1H NMR spectroscopy), thus
indicating that the nitro-containing multiple-release com-
pounds are only degraded on reduction of the nitro group.
Since the reported cascade-release dendrimers may be
useful for drug delivery, we have evaluated both multiple-
release monomers aminodiol 1 and aminotriol 12, which are
expected to be regenerated under physiological conditions
Received: May 21, 2003
Revised: July 10, 2003[Z51942]
Keywords: dendrimers · drug delivery · fragmentation ·
.
multiple release · prodrugs
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