The azaquinone-methide elimination: Comparison study of 1,6- and 1,4-eliminations under physiological conditions

Article abstract of DOI:10.1039/b808198k

The azaquinone-methide elimination is a powerful and efficient reaction useful for disassembly of spacers in prodrug systems. We and others have used the spacer-technique to develop dendritic and polymeric self-immolative molecular systems that can disass

Full text of DOI:10.1039/b808198k

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www.rsc.org/obc | Organic & Biomolecular Chemistry
The azaquinone-methide elimination: comparison study of 1,6- and
1,4-eliminations under physiological conditions†
Rotem Erez and Doron Shabat*
Received 14th May 2008, Accepted 12th June 2008
First published as an Advance Article on the web 23rd June 2008
DOI: 10.1039/b808198k
The azaquinone-methide elimination is a powerful and effi-
cient reaction useful for disassembly of spacers in prodrug
systems. We and others have used the spacer-technique to
develop dendritic and polymeric self-immolative molecular
systems that can disassemble through a domino-like mech-
anism upon a stimulus event. In this report, we study the
disassembly of a system that can disintegrate through para-
and ortho-azaquinone-methide eliminations. The disassembly
was evaluated with molecules that undergo single 1,6- or
1,4-elimination and with molecules that undergo double 1,6-
and 1,4-elimination. The 1,6-elimination was slightly faster
than the 1,4-elimination under physiological conditions. This
study sheds light on the disassembly-behavior of prodrug
Fig. 1 Activation mechanism of a tripartate prodrug by enzymatic
cleavage and elimination through an azaquinone-methide species.
systems.
In 1981, the group of Katzenellenbogen discovered that 4-amino-
benzyl alcohol can function as an efficient self-immolative spacer
in prodrug design. The spacer is used to link a drug to an
enzymatic substrate, thereby generating a tripartate prodrug.1 The
prodrug is stable as long as the enzymatic substrate is attached.
However, cleavage of the substrate by the enzyme generates an
intermediate that rapidly releases the free active drug. The concept
was demonstrated with 4-nitroaniline as a model drug and lysine
as an enzymatic substrate for trypsin (Fig. 1). Incubation of
compound 1 with trypsin resulted in release of amine 1a that
spontaneously underwent 1,6-elimination to generate azaquinone-
methide 1b and free 4-nitroaniline.
Since this discovery, numerous scientists have used this spacer-
technique for prodrug design.2 It was shown that 4-hydroxy-
benzyl alcohol and 4-mercapto-benzyl alcohol also undergo 1,6-
elimination upon exposure of the hydroxyl or the thiol groups.3,4
When 2-amino-benzyl alcohol is used as a spacer, the system can
undergo analogous 1,4-elimination through an ortho-azaquinone-
methide. We have used the spacer technique to develop dendritic
and polymeric self-immolative molecular systems that disassemble
through a domino-like mechanism upon a stimulus event.5–7 The
systems act as molecular amplifiers for diagnostic and drug
delivery applications.8,9 The azaquinone-methide elimination was
shown to serve as a powerful and efficient reaction in the design
of self-immolative molecular systems.10 The design of a self-
immolative dendritic adaptor was based on a molecule where
both 1,4 and 1,6-elimination reactions occurred in the same
aromatic ring (Fig. 2). In this contribution, we report a comparison
study of the reaction rates of para- and ortho-azaquinone-methide
eliminations. Compound type I (Fig. 2) has two reporter groups
(R) on the benzylic substituents, located at the para and ortho
positions of the corresponded aniline. An amide linkage with an
enzymatic substrate blocks the aromatic-amino group. Removal
of the substrate by the enzyme initiates the disassembly of the
system through para and ortho-azaquinone-methide eliminations
to release the two reporters.
In order to compare the rates of 1,4 and 1,6-elimination,
we initially synthesized two simple model compounds 2 and 3
(Fig. 3, see Supporting Information for synthesis details†). Both
compounds have a trigger group that can be removed through
cleavageofthe phenylacetamide catalyzed bypenicillin-G-amidase
(PGA).11 Enzymatic cleavage is followed by spontaneous 1,4- or
1,6-elimination and decarboxylation to release the corresponded
aniline (the 4-amino-benzyl alcohol unit is used as a spacer
between the aniline and the phenylacetamide enzymatic substrate).
While aniline 2a undergoes 1,6-elimination to release the 5-
amino-2-nitrobenzoic acid, aniline 3a disintegrates through 1,4-
elimination.
Compounds 2 and 3 were incubated in phosphate buffer saline
(pH 7.4; PBS) with PGA and the release of 5-amino-2-nitrobenzoic
acid was monitored by RP-HPLC (Fig. 4). Compound 2 disassem-
bled within 30 min without any detectable intermediate to release
the reporter unit (Fig. 4A). The disassembly of compound 3 under
the identical conditions occurred over 60 min and the appearance
and disappearance of intermediate 3a were observable (Fig. 4B).
These results show that the reaction-rate of the 1,4-elimination is
slightly slower than that of the 1,6-elimination (the identification of
Department of Organic Chemistry, School of Chemistry, Raymond and
Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv
69978, Israel. E-mail: chdoron@post.tau.ac.il; Fax: +972 (0) 3 640 9293;
Tel: +972 (0) 3 640 8340
† Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/b808198k
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Fig. 2 Disassembly of compound type I through 1,6- and 1,4-elimination reactions to release the reporter units.
Fig. 3 PGA-catalyzed disassembly of compound 2 through 1,6-elimination and of compound 3 through 1,4-elimination to release 5-amino-2-nitrobenzoic
acid.
intermediate 3a was confirmed through its UV spectrum observed
by the photo-diode array detector of the HPLC).
of compound 4 disappeared within 1 h. The reporter 5-amino-
2-nitrobenzoic acid, linked at the para-benzyl substituent, was
released faster than the ortho-linked 4-nitroaniline. For compound
5, the para-linked 4-nitroaniline unit was released more rapidly
than the 5-amino-2-nitrobenzoic acid (Fig. 6B). In both examples,
the disassembly was complete after approximately six hours. The
concentration of PGA used, 0.01 mg mL−1, allowed the enzymatic
cleavage on a time-scale suitable for monitoring of the reaction by
HPLC.
The results obtained in this study clearly indicate that the 1,6-
elimination through the para-azaquinone-methide occurs faster
than the 1,4-elimination through the ortho-azaquinone-methide
species. Although compound 2 disassembled rapidly through 1,6-
elimination with no detectable intermediate, the disassembly of
compound 3 through 1,4-elimination occurred more slowly and
the generation of intermediate 3a was observed. The disassembly
With these results in hand, we evaluated the disassembly of
compound type 1. Compounds 4 and 5 were synthesized (see
Supporting Information†) with a triggering group designed for
cleavage by PGA (Fig. 5). Upon activation, each of the compounds
can undergo 1,4 and 1,6-elimination reactions to release its
reporter units according to Fig. 1. Two different reporters were
introduced on each one of compounds 4 and 5. On compound 4,
the reporter 5-amino-2-nitrobenzoic acid was linked at the para-
benzyl substituent and the reporter 4-nitroaniline at the ortho
one. On compound 5, 4-nitroaniline was linked at the para-benzyl
substituent and 5-amino-2-nitrobenzoic acid at the ortho one.
Compounds 4 and 5 were incubated in PBS (pH 7.4) with PGA
and the release of 5-amino-2-nitrobenzoic acid and 4-nitroaniline
was monitored by RP-HPLC (Fig. 6). As shown in Fig. 6A, most
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Fig. 4 PGA-catalyzed the release of 5-amino-2-nitrobenzoic acid from
compounds 2 and 3 through 1,6- and 1,4-eliminations respectively.
Conditions: 2 or 3, [500 lM] in PBS 7.4 and PGA [0.01 mg mL−1].
Fig. 6 PGA-catalyzed the release of 5-amino-2-nitrobenzoic acid and
4-nitroaniline from compounds 4 and 5 through 1,6- and 1,4-eliminations.
Conditions: 4 or 5, [500 lM] in PBS 7.4, PGA [0.01 mg mL−1].
of compounds 4 and 5, which bear two different substituents to
allow independent analysis of 1,4- and 1,6-elimination reactions
of the same compound, occurred with a faster release of the para-
reporter than the ortho one, indicating that the 1,6-elimination
was faster. However, the difference between the reaction rates of
the two eliminations was relatively small. The biphasic kinetics
observed for the disassembly of compounds 4 and 5, is probably
derived of the mechanism described in Fig. 2. Both the para- and
ortho-azaquinone-methide eliminations can be used to effectively
release drug/reporter molecules under physiological conditions. A
similar observation was made previously with mercapto analogs
of the corresponding anilines used in this report.3
In summary, we have described here a study of a new system
that can disassemble through para- and ortho-azaquinone-methide
elimination.12 The disassembly was evaluated with molecules
that undergo single 1,6- or 1,4-elimination and with molecules
that undergo double-elimination. The 1,6-elimination was shown
to occur slightly faster than the 1,4-elimination under physi-
ological conditions. This study will aid in design of prodrug
systems that are based on a stimulus activation and release
mechanism.
Fig. 5 PGA-catalyzed the release of 5-amino-2-nitrobenzoic acid and 4-nitroaniline from compounds 4 and 5 through a 1,6- and 1,4-double elimination.
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Acknowledgements
D.S. thanks the Israel Science Foundation (ISF) and the Binational
Science Foundation (BSF) for financial support.
Notes and references
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12 While this manuscript was under preparation, a related study, de-
scribing a similar system that undergoes 1,6- and 1,4-elimination
reactions, was published. However, unlike our, the system analyzed by
Warnecke et al. was separately activated with a chemical reagent and
solubility problems did not allow evaluation of the disassembly under
physiological conditions. A. Warnecke and F. Kratz, J. Org. Chem.,
2008, 73, 1546.
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The Royal Society of Chemistry 2008
©