Versatile approach to α-alkoxy carbamate synthesis and stimulus-responsive alcohol release

Article abstract of DOI:10.1039/c2ob26571k

A series of α-alkoxy carbamates that cleave under mild conditions to release alcohols has been synthesized through a multicomponent process. The relationship between structural features in these compounds and the rate of alcohol release in the presence of basic hydrogen peroxide has been studied. The preparation of carbamates that cleave under other conditions has been demonstrated.

Full text of DOI:10.1039/c2ob26571k

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PAPER
Versatile approach to α-alkoxy carbamate synthesis and stimulus-responsive
alcohol release†
R. Adam Mosey and Paul E. Floreancig*
Received 9th July 2012, Accepted 14th August 2012
DOI: 10.1039/c2ob26571k
A series of α-alkoxy carbamates that cleave under mild conditions to release alcohols has been
synthesized through a multicomponent process. The relationship between structural features in these
compounds and the rate of alcohol release in the presence of basic hydrogen peroxide has been studied.
The preparation of carbamates that cleave under other conditions has been demonstrated.
Introduction
Stimulus-responsive cleavage reactions have found broad use for
applications in temporally-controlled drug release, chemical
sensing, and material decomposition processes.1 Carbamates
have frequently been used to release amines in these processes
due to the diverse array of stimuli that can be employed for the
decomposition step. We have initiated a program by which
α-alkoxy carbamate cleavage can serve to release alcohols in
response to externally-applied stimuli, as shown in Scheme 1.
The capacity to direct drug release at a particular location
Fig. 1 Schematic of a cargo-releasing composite structure that contains
localization and triggering groups.
demonstrate the merits of the multicomponent reaction for the
preparation of these structures.
would be a significant attribute for a drug-delivery agent. The
benefit of employing α-alkoxy carbamates as delivery agents2 is
that the carbamate structure contains a branch that can be used to
append cell-targeting agents. Thus a single molecule can contain
directing, triggering, and cargo groups (Fig. 1). This design has
been implemented in elegant studies from the Shabat,3 Shin,4
and Sinha5 groups. These successful constructs highlight the
utility of the design yet reveal that enhanced synthetic accessibil-
ity would be desirable for rapid property optimization. In this
manuscript we demonstrate that several α-alkoxy carbamates can
be prepared rapidly through a multicomponent reaction and illus-
trate the manner in which this protocol can be used to access
compounds that release alcohols at varying rates. Compounds
with different triggering mechanisms have been prepared to
Results and discussion
Our approach to α-alkoxy carbamate synthesis employed a
sequence of nitrile hydrozirconation, acylation, and alcohol
addition.6 This protocol has successfully been applied to rapid
syntheses of cyclic amides,7 the natural products pederin and
psymberin, and many analogs.8 Conducting the multicomponent
reaction at a late stage in the synthetic sequence provides abun-
dant opportunities for optimizing molecular properties through
structural diversification.
The synthesis of a representative cleavable α-alkoxy carba-
mate is shown in Scheme 2. We chose to explore peroxide-trig-
gered compounds in consideration of the potential for drug
delivery to oxidant-rich medical conditions such as cancer9 and
neurodegeneration.10 Our design was based on Chang’s per-
oxide-released fluorophores11 in which boronate oxidation forms
a phenoxide that releases its cargo through p-quinone methide
formation.12 Thus, known benzylic alcohol 1 was treated with a
solution of phosgene in toluene to form chloroformate 2. Hydro-
Scheme 1 Stimulus-promoted cleavage of α-alkoxy carbamates.
t
Department of Chemistry, University of Pittsburgh, Pittsburgh,
Pennsylvania 15260, USA. E-mail: florean@pitt.edu;
Fax: +1 412-624-8626; Tel: +1 412-624-8727
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26571k
zirconation of BuCN (3) with Cp2Zr(H)Cl followed by the
addition of freshly-prepared 2 yielded intermediate acylimine 4.
The synthesis was completed through the addition of neopentyl
alcohol to provide 5 in 64% yield.
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Fig. 2 Time dependent release of neopentyl alcohol from 5. The AB
pattern arises from 5 and the growing singlet arises from neopentyl
alcohol. See Scheme 3 for experimental conditions.
Scheme 2 Multicomponent approach to α-alkoxy carbamate synthesis.
1
neopentyl alcohol was approximately 60% as determined by H
NMR, but subsequent experiments that released less volatile
alcohols showed that isolated yields are much higher (see
below), suggesting that yield determination can be influenced by
relaxation rate differences in comparison to the internal standard.
Therefore determinations of the half lives for product formation
provides a valid comparison for determining the relative reactiv-
ities of different analogs. We used the conversion at 35 min as
the end point for t1/2 determinations. Using conversion levels at
later times does not significantly change the values. The half life
for the release of neopentyl alcohol from 5 was 12 min.
This facile approach to α-alkoxy carbamate synthesis allowed
us to prepare a number of analogous structures (Fig. 3)13 in
accord with our objective of determining the influence of struc-
tural perturbations in the triggering group, the nitrile group, and
the alcohol on the rate of peroxide-mediated alcohol release.
Steric hindrance around the leaving group was altered by prepar-
ing 10 from acetonitrile, while the electronics of the nitrile com-
ponent were altered by synthesizing 11 and 12 from the
corresponding electron-poor and electron-rich aromatic nitriles.
The carbamate group was altered through the incorporation of a
fluorine, a methoxy group, and a benzylic methyl branch
(13–15). Carbamate 16 was prepared to study the impact of
increasing the leaving group ability of the cargo. The selection
of alcohols in these analogs was based on the presence of
Scheme 3 Peroxide-mediated alcohol release. [5]0 = 6 mM, [H2O2]0 =
90 mM, T = 300 K.
The breakdown of compound 5 was effected by treatment
with urea·H2O2 at pH = 8.0 (phosphate buffer) in a mixture of
CD3CN and D2O (Scheme 3). This allowed for direct monitoring
1
of starting material consumption and product formation by H
NMR. This experiment showed that the oxidation of the boronate
to phenoxide 6 was rapid, as indicated by the subtle change in
the chemical shifts of the diastereotopic hydrogens of the neo-
pentyl ether group after 1 min (Fig. 2). Acyl aminal breakdown
proceeded through quinone methide loss to form 7, decarboxyla-
tion to yield 8, and aminal breakdown to yield neopentyl alcohol
and imine 9. The imine was not observed in this reaction due to
its rapid reaction with water to yield the corresponding aldehyde.
Aldehydes oxidize rapidly with basic hydrogen peroxide,
making the loss of this component difficult to monitor in this
experiment. Under different conditions, however, this decompo-
sition process could be used to release alcohol and aldehyde
cargo through a single triggering event. Alcohol release was
slower than phenoxide formation, indicating that oxidation is not
the rate-determining step when high peroxide concentrations are
employed for the breakdown.
1
methylene groups that appear as AB quartets in the H NMR
spectra of the carbamates then collapse to singlets upon release,
thereby allowing decomposition to be monitored readily. Vinyl
boronate 17 was prepared from the corresponding allylic chloro-
formate to determine the relative merits of utilizing the β-elimin-
ation of an enolate intermediate14 to promote acyl aminal
breakdown. Benzyl carbamate 18 was synthesized as a control
compound to verify that alcohol release can be attributed to bor-
onate oxidation.
The versatility of the synthetic protocol was further probed
through the preparation of a number of additional structures that
contain different triggering groups. Acetoxy carbamate 19 was
prepared for cargo release in response to esterase-mediated clea-
vage. Azido carbamate 20 was prepared for cargo release in
thioredoxin-rich environments. Nitro-substituted carbamate 21
Neopentyl alcohol was released at a similar rate to the
decomposition of the carbamate (Fig. 2), as determined by inte-
grating the growing methylene singlet from the alcohol and the
AB system from the carbamate and comparing the intensities to
THF, which was added as an internal standard. The yield of
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Fig. 4 Comparison of the rates of alcohol release from structurally-
varied nitrile precursors. See Scheme 3 for experimental conditions.
Experiments were run in triplicate.
Fig. 3 Structurally-varied carbamates.
was prepared for cargo release in the presence of nitrido
reductase. The latter two compounds were designed for release
in hypoxic environments that are associated with many forms of
cancer.15
Fig. 5 Impact of carbamate variation on alcohol release. See Scheme 3
for experimental conditions. Experiments were run in triplicate.
We exposed compounds 10, 11, and 12 to the oxidative frag-
mentation conditions to determine the influence of the group on
the nitrile fragment on the rate of alcohol release. As shown in
Fig. 4 very little difference was observed for these compounds,
with 10 showing a t1/2 of 9 min, 11 showing a t1/2 of 12 min, and
12 showing a t1/2 of 9 min. The similarities of these numbers
contrasts with the significant steric and electronic differences
between the structures and demonstrates that the rate determining
step in this process is not the release of the neopentyl alcohol
from a tetrahedral intermediate similar to 8. The stability of
control compound 18 toward oxidative fragmentation is also
confirmed in Fig. 4. This demonstrates that the nitrile-derived
subunit of these structures can be selected to facilitate the syn-
thesis or to optimize a different property without consequence
on the release rate.
addition of a methoxy group to the arene provided a substantial
rate increase, with a t1/2 for 14 being 2 min. The addition of a
methyl group at the benzylic site resulted in a noticeable rate
increase, with a t1/2 of 6 min for 15. These results are consistent
with benzyl cleavage being the rate determining step in this
process.16 The addition of a branching alkyl group at the
benzylic position and the incorporation of an electron-donating
methoxy group to the arene facilitate the departure of the car-
boxyl group. These observations show that factors that stabilize
a benzylic cation will promote breakdown. The negligible
impact of incorporating a fluorine into the carbamate is therefore
somewhat surprising since this substitution should inductively
blunt the electron-donating capacity of the phenol that forms
from boronate oxidation. We propose that this inductive stabiliz-
ation can be balanced by the higher concentration of the more
reactive phenoxide that results from the lower pKa of the fluori-
nated analog. The decomposition rates for 5 and 14 did not vary
The next phase of this project was to determine the impact of
perturbations to the alkoxy group of the carbamate on the rate of
alcohol release. The peroxide-mediated breakdowns of 13, 14,
and 15 were compared to 5 (Fig. 5). While the incorporation of a
fluorine atom to the benzene ring did not result in a significant
change in the rate of alcohol release (t1/2 for 13 = 11 min), the
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Fig. 6 Release as a function of alcohol structure. See Scheme 3 for
experimental conditions. Experiments were run in triplicate.
Fig. 7 Release from the allylic carbamate. See Scheme 3 for experi-
mental conditions. Experiments were run in triplicate.
significantly when the substrate concentration was 3 mM and
[H2O2] was 30 mM (see ESI†).
The influence of alcohol nucleofugacity on the cleavage rate
was determined by comparing the rates of peroxide-mediated
breakdowns of 5 and aryl ether 16. These processes were shown
to proceed with essentially identical rates (t1/2 = 10 min for 16),
as shown in Fig. 6, despite the greater stability of the phenoxide
leaving group in comparison to an alkoxide leaving group. This
outcome is consistent with the hypothesis that the facility of
benzylic carbon–oxygen bond cleavage, and not tetrahedral
intermediate breakdown, determines the rate of alcohol release.
The decomposition of 16 appears to proceed in higher yield than
the decomposition of 5, though this can be attributed to a differ-
ence in 1H relaxation rates in these NMR-derived plots.
Scheme 4 Determination of an isolated yield.
Allylic carbamate 17 differs from the previous compounds in
that its peroxide-mediated oxidation forms an enolate rather than
a phenoxide. β-Elimination leads to cargo release and the for-
mation of acrolein. The breakdown of this compound, in com-
parison to rapidly reacting substrate 14 and parent structure 5, is
shown in Fig. 7. This process is extremely fast, with a t1/2 of
<2 min, showing that this structural variant will be useful for
applications where extremely rapid cargo release is desirable. We
prepared carbamate 22 in an effort to determine the yield of the
released alcohol.13 The alkoxy group in 22 was selected to be
sufficiently non-volatile and UV-active to facilitate isolation.
Exposing 22 to the standard cleavage conditions (Scheme 4) for
1 h provided alcohol 23 in an 82% isolated yield, demonstrating
1
that cargo release proceeds more effectively than the H NMR
experiments would indicate.
This method was applied to the preparation of a compound
that is designed to deliver an anti-oxidant to mitochondria17 in
an effort to prepare agents that mitigate radiation damage by sup-
pressing the formation of reactive oxygen species.18 The design
of the compound and its synthesis are shown in Scheme 5. The
boronate carbamate group was employed to trigger compound
release in the presence of the high peroxide concentrations in
mitochondria.19 Pentamethyl chromanol (PMC, 24) was selected
for release due to its ability to mitigate the effects of cytochrome
c-mediated mitochondrial damage.20 The triphenylphosphonium
Scheme 5 Application to the synthesis of a potential mitigator of radi-
ation damage.
ion was included to promote passage through mitochondrial
membranes.21 Hydrozirconation of nitrile 25 followed by acyla-
tion with 2 and nucleophilic addition of 24 provided carbamate
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the rapid preparation of structurally diverse α-alkoxy carbamates.
These carbamates have been designed to release the alcohol
component in response to a precise chemical signal as an entry
into the development of drug delivery vehicles. The utility of
this approach has been demonstrated in a quick study of the
relationship between structural features and the rate of peroxide-
mediated alcohol release from boronate-substituted benzylic
carbamates. This work showed that the release rate is controlled
by the rate of quinone methide formation rather than boronate
oxidation or tetrahedral intermediate collapse. Carbamates that
cleave in the presence of nucleophiles and reducing agents have
also been prepared to highlight the generality of the pathway. A
complex that contains a triphenyl phosphonium ion for mito-
chondrial transport, a boronate-substituted benzylic carbamate as
a peroxide-mediated trigger, and a chromanol derivative has
been prepared for the delivery of anti-oxidants for studies on the
directed mitigation of damage from reactive oxygen species. The
ability of 28 and related structures to act as radiation mitigators
is currently under investigation and the results will be reported
elsewhere. We anticipate that the facile preparation of structurally
diverse agents that can release cargo under defined conditions
will prove to be useful for applications in targeted drug delivery
and for controlled tailoring of materials.
Fig. 8 PMC release from 28 in response to variations in pH and H2O2
concentration. Reactions were performed using 40 μM of 28 or 29 in
95% aqueous phosphate buffer/5% EtOH at 37 °C. Experiments were
run in triplicate.
26 in 58% yield. Removal of the silyl group followed by acyla-
tion with commercially available acid 27 provided 28 in good
yield. The rapid synthesis of this compound shows that a more
stable linkage to the phosphonium ion could be incorporated if
the ester linkage is susceptible to esterase cleavage in cellular
studies. Benzyl carbamate 29 was prepared as a control com-
pound for comparison in breakdown studies.
Acknowledgements
This work was supported by generous funding from the National
Science Foundation (CHE-0848299) and the National Institutes
of Health (AI068021). We thank Ms Stephanie Garrell and
Mr Akira Shimizu (recipient of a Brackenridge undergraduate
research fellowship) for assistance in chloroformate synthesis.
We thank Dr Detcho Stoyanovsky (Department of Occupational
Health and Safety, University of Pittsburgh) for helpful discus-
sions and technical assistance.
The peroxide-mediated breakdown of 28 to release PMC was
studied in aqueous media (phosphate buffer containing 5%
EtOH) at pH 7.2 and 8.0 (the pH of the mitochondrial matrix).22
These studies were performed with micromolar concentrations of
the carbamate and H2O2 at 37 °C to mimic biological conditions.
These concentrations required that the reactions be monitored by
HPLC using an electrochemical detector. Treating 28 (40 μM)
with varying concentrations of H2O2 (10, 20, and 40 μM)
resulted in the liberation of PMC in a dose-dependant manner
(Fig. 8). The release rate was notably accelerated when the reac-
tion was performed at higher pH. A pH = 8.0 reaction mixture
containing 40 μM of 28 and H2O2 generated approximately
20 μM of PMC within 30 min, whereas the reaction at pH = 7.2
required 90 min to reach a similar concentration. The necessity
of the boronate ester for carbamate breakdown and alcohol
release was also demonstrated as PMC accumulation was not
observed following exposure of 29 to H2O2 and the small
amounts of PMC that were detected were not distinguishable
from noise. The release of PMC from 28 proceeded much more
slowly than the release of neopentyl alcohol from 5. This can be
attributed to the rate reduction for boronate oxidation that results
from the low concentrations. The rate of alcohol release is depen-
dent on the rate of quinone methide formation, and the rate of
quinone methide formation is dependent upon the concentration
of the phenoxide intermediate that forms through a bimolecular
reaction between the substrate and the hydroperoxide anion,
thereby explaining the low release rates in these reactions.
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