Photolytic release of butyric acid from oxygen-and nitrogen-based heteroaromatic cages

Article abstract of DOI:10.1002/ejoc.201101392

In order to develop butyric acid photoactive prodrugs, new heteroaromatic conjugates based on oxygen and nitrogen were synthesised and evaluated under irradiation at 254, 300, 350 and 419 nm. Light-triggered uncaging of butyric acid from the corresponding heterocyclic cages was achieved with complete release of the drug in short times. Naphtho[2,3-d]oxazole, naphtho[1,2-d] oxazole, 3-oxo-3H-benzo[f]benzopyran, 2-oxo-2H-benzo[h]benzopyran and 6-oxo-6H-benzopyrano[6,7-d]oxazole conjugates were readily photolysed, and the best results were obtained for naphtho-oxazoles at 254 and 300 nm and for 3-oxo-3H-benzo[f]benzopyran, 2-oxo-2H-benzo[h]benzopyran and 2-methyl-6-oxo-6H-benzopyrano[6,7-d]oxazole at 350 nm. 3-Oxo-3H-benzo[f] benzopyran also afforded good results at 419 nm. The photophysical processes involved were further elucidated by the use of time-resolved fluorescence techniques. Butyric acid photoactive prodrugs based on oxygen and nitrogen heteroaromatic conjugates were synthesised and evaluated by irradiation at 254, 300, 350 and 419 nm. The photophysical processes involved were further elucidated by the use of time-resolved fluorescence techniques. Copyright

Full text of DOI:10.1002/ejoc.201101392

FULL PAPER
DOI: 10.1002/ejoc.201101392
Photolytic Release of Butyric Acid from Oxygen- and Nitrogen-Based
Heteroaromatic Cages
[a]
[a]
[b]
[a]
Ana M. S. Soares, Ana M. Piloto, Graham Hungerford, Susana P. G. Costa, and
M. Sameiro T. Gonçalves*[
a]
Keywords: Prodrugs / Butyric acid / Oxazoles / Oxobenzopyran / Coumarin / Photolysis / Fluorescence spectroscopy
In order to develop butyric acid photoactive prodrugs, new
heteroaromatic conjugates based on oxygen and nitrogen
were synthesised and evaluated under irradiation at 254,
benzopyrano[6,7-d]oxazole conjugates were readily pho-
tolysed, and the best results were obtained for naphtho-
oxazoles at 254 and 300 nm and for 3-oxo-3H-benzo[f]benzo-
pyran, 2-oxo-2H-benzo[h]benzopyran and 2-methyl-6-oxo-
6H-benzopyrano[6,7-d]oxazole at 350 nm. 3-Oxo-3H-benzo-
[f]benzopyran also afforded good results at 419 nm. The pho-
tophysical processes involved were further elucidated by the
use of time-resolved fluorescence techniques.
300, 350 and 419 nm. Light-triggered uncaging of butyric
acid from the corresponding heterocyclic cages was achieved
with complete release of the drug in short times. Naphtho-
[2,3-d]oxazole, naphtho[1,2-d]oxazole, 3-oxo-3H-benzo[f]-
benzopyran, 2-oxo-2H-benzo[h]benzopyran and 6-oxo-6H-
Introduction
involved in the regulatory mechanisms for gene expression
Several strategies for the development of pharmacologi-
cally inert chemical derivatives, prodrugs, which can be con-
verted into active drug molecules, have been explored to
overcome common drawbacks such as low oral drug ab-
sorption, lack of site specificity, chemical instability, toxicity
and poor patient acceptance.[1–3] Photoactive prodrugs with
a suitable photolabile group, whose reactivity can be con-
trolled by selecting the wavelength of the excitation light,
could be an alternative to the molecular design of prodrugs.
The timed and accurate delivery of a drug at a specific loca-
tion in vivo or in vitro presents a challenging goal that can
be achieved by using photoactive prodrugs that are able to
decompose rapidly upon photoirradiation. In recent reports
in the field of prodrugs, there have been some examples of
light as the triggering agent for the optimisation of drug
known to promote markers of cell differentiation, apoptosis
[7,8]
and cell-growth control.
Prodrugs based on butyric acid,
upon intracellular hydrolytic degradation, release acids and
aldehydes, modulate gene expression, and induce histone
hyperacetylation, differentiation and apoptosis of cancer
cells. Prodrugs that release formaldehyde increase apoptosis
of cancer cells, at concentrations about 10-fold lower than
butyric acid and 100 times faster. The formaldehyde re-
leased from these prodrugs has been shown to be a critical
antiproliferative factor that induces differentiation and cell
[8–11]
death.
Our recent research has focussed on the synthesis and
application of new oxygen and nitrogen heterocycles as
photosensitive groups for the protection of carboxylic acid
[12–18]
and amino groups.
In order to extend the scope of
[
4–6]
delivery.
Given the promising results obtained, this
applications of such groups to drug-delivery research in the
form of photoactive prodrugs, this work evaluates the use
of naphtho[2,3-d]oxazole, naphtho[1,2-d]oxazole, 3-oxo-3H-
benzo[f]benzopyran, 2-oxo-2H-benzo[h]benzopyran and 6-
oxo-6H-benzopyrano[6,7-d]oxazoles in the light-triggered
release of butyric acid from the corresponding heterocyclic
cages. The stability to irradiation of the ester bond between
butyric acid and the caging group has been evaluated in a
photochemical reactor at 254, 300, 350 and 419 nm, and
uncaging data have been acquired. The photophysical pro-
cesses involved have been further elucidated by the use of
strategy could allow for a more patient-friendly drug deliv-
ery and availability scheme, which involves less complex
dosing schedules and a lower potential for side effects.
Drugs that contain ionisable polar groups such as
carboxylic acids are poorly absorbed from the gastrointesti-
nal tract due to lipophilicity/solubility issues. The absorp-
tion can be improved by masking the carboxyl group by
[
1]
forming derivatives through different covalent linkages.
Butyric acid is a naturally occurring short-chain fatty acid
[
[
a] Centre of Chemistry, University of Minho, Campus of Gualtar, time-resolved fluorescence techniques.
4710-057 Braga, Portugal
Fax: +351-253604382
E-mail: msameiro@quimica.uminho.pt
b] HORIBA Jobin Yvon IBH Ltd,
Results and Discussion
4
5 Finnieston Street, Glasgow G3 8JU, UK
The synthesis of bromomethylated naphtho[1,2-d]ox-
azole (2) was achieved by a condensation reaction between
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101392.
922
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 922–930

Photolytic Release of Butyric Acid from Heteroaromatic Cages
1
-aminonaphthalen-2-ol and bromoethanoic acid mediated
[
16]
by polyphosphoric acid according to a known procedure.
We have previously reported the syntheses of naphtho[2,3-
d]oxazole (1), 3-oxo-3H-benzo[f]benzopyran (3), 2-oxo-2H-
benzo[h]benzopyran (4) and 6-oxo-6H-benzopyrano[6,7-d]-
[
12,16,17]
oxazoles 5 and 6.
Compounds 1–6 were used in the derivatisation of
butyric acid in the presence of potassium fluoride (for 1–3,
5
and 6) or N,NЈ-dicyclohexylcarbodiimide (DCC)/1-hy-
droxybenzotriazole (HOBt, for 4) in N,N-dimethylform-
amide (DMF) at room temperature,[
14,19]
which resulted in
the ester prodrugs 7–12 in good yields (Table 1, Scheme 1).
All compounds synthesised were fully characterised by
1
13
HRMS, IR, H and C NMR spectroscopy. The IR spectra
of 7–12 showed bands attributable to the stretching vi-
–1
brations of the ester carbonyl group from 1718 to1749 cm .
1
H NMR spectra showed signals of the butyric acid moiety,
the methyl (δ = 0.93–1.02 ppm) and two methylene groups
(
δ ≈ 1.72 and 2.49 ppm). The heterocyclic methylene groups
were also visible for all compounds (δ = 4.14–5.69 ppm).
The confirmation of the presence of the newly formed ester
1
3
linkages was also supported by C NMR spectral signals
of the carbonyl group, which were found at δ = 172.68–
173.59 ppm.
UV/Vis spectroscopic characterisation was carried out to
obtain the parameters needed for monitoring the uncaging
–
5
process. Absorption spectra of degassed 10 m solutions in
absolute ethanol and in a methanol/4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer (80:20) solu-
tion of 7–12 were measured and compared to those of pre-
cursors 1–6. Absorption maxima and molar absorptivities
are reported in Table 1. By comparison of the absorption
maxima for all compounds in both solvents, no significant
changes were observed. The differences between the pairs Scheme 1. Synthesis of butyric acid prodrugs 7–12. (a) KF/DMF,
room temp.; (b) DCC/HOBt, DMF, room temp.
of compounds were examined. For the naphtho-oxazole
compounds 7 and 8, the latter has a more defined band
structure and an absorption maximum ca. 20 nm redshifted ment of the benzene ring in 7 by an oxopyran in 12 also
compared to that of 7. A similar bathochromic shift was resulted in a comparable absorption redshift.
seen between oxobenzopyrans 9 and 10, and no difference
The fluorescence spectra were also measured as the use
was found between 11 and 12, which incorporate an oxo- of fluorescent labelling with its increased sensitivity over
benzopyran chromophore fused with an oxazole. Replace- UV/Vis absorption techniques makes it suitable for analyti-
Table 1. Yields, UV/Vis absorption and emission data in absolute ethanol and methanol/HEPES buffer (80:20) solutions for 1–12.
Yield
%]
Ethanol
Methanol/HEPES (80:20)
[
a]
[a]
Δλ[b]
[a]
abs
[a]
Δλ[b]
[
λ
abs
logε
λ
em
Φ
F
λ
logε
λ
em
Φ
F
1[c]
2
13
98
83
47
34
23
97
89
80
30
95
47
334
326
354
365
326
325
300
322
350
375
325
322
3.67
3.94
4.10
3.69
3.98
3.80
3.52
3.90
3.92
3.88
3.96
3.93
380
355
472
460
404
424
383
355
466
474
425
457
0.28
0.22
0.03
0.45
0.06
0.01
0.42
0.11
0.02
0.38
0.11
0.03
3624
2506
7062
5658
5922
7184
7224
2887
7112
5570
7240
9174
334
310
355
371
325
325
302
321
352
375
326
321
3.60
4.61
3.47
3.73
3.94
3.54
3.99
4.26
4.22
3.85
3.83
4.14
380
354
458
469
397
396
355
355
484
482
424
457
0.02
0.01
0.10
0.45
0.005
0.001
0.03
0.21
0.46
0.28
0.10
0.019
3624
4009
6335
5632
5580
5517
4944
2984
7748
5920
7090
9271
[
[
[
[
d]
e]
c]
c]
3
4
5
6
7
8
9
1
1
1
0
1
2
–
1
[16]
[d] Yield and absorption data in ethanol from ref.[21] [e] Yield and
[
a] In nm. [b] In cm . [c] Yield and absorption data from ref.
[17]
absorption data from ref.
Eur. J. Org. Chem. 2012, 922–930
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
923

M. S. T. Gonçalves et al.
FULL PAPER
cal purposes. The outcome of these steady-state measure-
ments is also given in Table 1. In recent years, the direction
of the improvement of photoreleasable groups has been
towards the application of polycyclic aromatic structures,
which are fluorophores in most cases. However, there can
be the added complication of fluorescence deactivation in
some photochemical processes.
Molecules of biological importance such as butyric acid
are not easily detected by UV/Vis absorption measurements
and have no or low intrinsic fluorescence, and thus the in-
troduction of a fluorophore is an appropriate strategy to
enhance the photophysical properties of the resulting conju-
gates and to provide a means to monitor photoinduced pro-
cesses. By considering the obtained values for the relative
fluorescence quantum yields (Φ ) for 7–12 in absolute etha-
F
nol and methanol/HEPES buffer (80:20) solution deter-
mined with 9,10-diphenylanthracene in ethanol as stan-
[
20]
dard, it is evident that the processes in these compounds
can be monitored by fluorescence techniques. The Stokes’
shifts were in the range 2506–9174 and 2984–9271 cm–1 in
ethanol and methanol/HEPES buffer (80:20) solution,
respectively, which is an advantageous property in fluo-
rescence techniques as it will minimise self-quenching
phenomena.
The photophysics of the different fluorophores in 7–12
in methanol/HEPES buffer (80:20) were investigated to help
elucidate the processes involved upon light irradiation.
Time-resolved fluorescence decays were measured at one
emission wavelength (Figure 1).
Figure 1 shows the different decay behaviour for the dif-
ferent sets of fluorophores 7–12. Compounds 7 and 8 exhi-
bit quite complex kinetic behaviour, which is indicative of
the presence of several excited states. The other compounds
appear to decay in a simpler manner, although the emission
from 12 is highly quenched, which is in keeping with the
low quantum yield given in Table 1. The associated lifetime
values extracted from the analysis of these decay curves
generally reflect the trend in the quantum yields (longer life-
times with higher quantum yields, Table 2).
To provide further insight into the processes that could
be occurring in the excited state, time-resolved fluorescence
measurements that monitored the emission at different
wavelengths were performed to allow time-resolved emis-
sion spectra (TRES) and, by the use of global analysis, de-
cay associated spectra (DAS) were calculated. Because of
the nature of time-correlated single-photon counting, which
uses very modest excitation energies (pJ per pulse), it was Figure 1. Fluorescence decay curves for 7–12. The instrumental re-
sponse function (IRF) is also shown.
not envisaged that significant photocleavage would occur
during the measurements. TRES for all compounds, except
9
and 10 (which bear an angular coumarin, 3-oxo-3H-
Upon photolysis, it was expected that an ion pair could
benzo[f]benzopyran and 2-oxo-2H-benzo[h]benzopyran, form, which would either undergo recombination to the es-
[
22]
respectively), showed changes in the spectral shape with ter or photocleavage.
Knowledge of the fluorescent spe-
time after excitation (Supporting Information) that hint at cies present and their kinetics may allow the rate of the
the presence of several species. This is in keeping with the photocleavage reaction to be estimated. The DAS can be
simple time-resolved fluorescence measurements (Figure 1 useful to provide a framework to assist in this determi-
and Table 2). The spectral shape of 9 and 10 appeared rela- nation by providing both spectral and dynamic infor-
tively constant, consistent with their simpler decay kinetic mation. The DAS for 7–12 are shown in Figure 2 and were
dominant decays of 7.4 and 9.4 ns, respectively.
obtained from the global analysis of the time-resolved de-
924
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 922–930

Photolytic Release of Butyric Acid from Heteroaromatic Cages
Table 2. Fluorescence lifetimes (τ) for 7–12 obtained from reconvolution analysis of the data presented in Figure 1.[a]
Lifetime [ns]
Relative amplitude [%]
τ
1
τ
2
τ
3
τ
4
f
1
f
2
f
3
f
4
7
8
9
1
1
1
8.04Ϯ0.48
6.58Ϯ0.06
7.41Ϯ0.02
9.41Ϯ0.02
5.47Ϯ0.31
1.93Ϯ0.15
3.13Ϯ0.06
1.44Ϯ0.01
3.68Ϯ0.12
–
2.12Ϯ0.01
0.02Ϯ0.01
0.80Ϯ0.02
–
–
–
0.11Ϯ0.01
–
0.052Ϯ0.009
9
23
16
4
–
87
98
23
–
–
–
9
45
–
–
–
–
–
–
–
–
–
84
96
100
4
2
n
0
1
2
–
–
[
a] The curves were fitted to a sum of exponentials of the form I(t) = ͚i
i i i
α τ , where α are normalised preexponential factors and the
relative amplitude or factional (f ) is given by f = (α ).
i
i
i
τ
i
)/(͚i
i i
α τ
cays taken from the TRES measurements with decays mea- presence of a very short-lived decay component (and associ-
sured at 5 nm intervals. Consistent with the TRES, the be- ated spectrum, because of the recovery of a clear, sensible
haviour of 9 and 10 appear slightly different with their emis- spectral shape it is unlikely that scattered light is the origin
sion dominated by the deexcitation of one excited state. In of this decay component; a fitting artefact, although un-
the case of the other compounds, global analysis shows the likely as the datasets consist of Ͼ 40 decays and the spectral
Figure 2. Normalised DAS for 7–12 in methanol/HEPES buffer (80:20) solution.
Eur. J. Org. Chem. 2012, 922–930
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
925

M. S. T. Gonçalves et al.
FULL PAPER
shape appears sensible, cannot be completely ruled out). tion. The plots of peak area (A) of the starting material vs.
The actual value of the shortest-lived decay should be irradiation time (t ) were obtained for each compound at
irr
treated with caution as it is at or beyond the resolution of the considered wavelengths. Peak areas were determined by
the equipment and hence ought to be considered as a very HPLC, which revealed a gradual decrease with time, and
short-lived decay component. The complexity of the decay were the average of three runs. The determined irradiation
behaviour of 7 and 8 may be indicative that these com- time represents the time necessary for the consumption of
pounds undergo photoinduced processes more readily than the starting materials until less than 5% of the initial area
the others, which can be seen later in the photolysis data was detected. Based on HPLC data for each compound,
and offers the possibility to monitor the whole uncaging the plot of lnA vs. tirr showed a linear correlation for the
process. The complete and rigorous attribution of the spec- disappearance of the starting material, which suggests a
tra and associated decay kinetics require the synthesis of first-order reaction and was obtained by the linear least-
appropriate model compounds for each of the chromo- squares method for a straight line with good correlation
phores. Because of the complexity seen in the fluorescence coefficients. The corresponding rate constants (k) were cal-
emission it can only be speculated that the shorter-lived spe- culated and are presented in Table 3.
cies are those initially formed with longer lifetimes associ-
ated with the products. The complete attribution of the
spectra will be left for a further study, and the results pre-
sented here demonstrate the ability of this technique to elu-
Table 3. Values of tirr [min] and k [ϫ10–2 min ] for the complete
–1
photolysis (Ͼ95%) of 7–12 at different wavelengths in methanol/
HEPES buffer (80:20) solution.
2
54 nm
300 nm
350 nm
t
cidate information.
t
irr
k
t
irr
k
irr
k
Compounds 11 and 12 exhibit higher energy (shorter
wavelength) spectra associated with a very fast decay and
longer-lived species at longer wavelengths. This can be in-
dicative of the longer-lived species formed from the initial
7
8
9
1
4.9
0.1
178
107
45
65.22
2918
1.70
2.86
6.59
3.95
5.6
0.8
253
170
33
53.42
400
1.19
2.86
6.59
3.16
Ͼ 2500
519
263
96
285
–
0.58
1.14
3.15
1.04
–
0
formation of the species associated with the shorter-lived 11
1
2
76
94
Ͼ 2500
fluorescence. In the case of 11, a further spectrum (ca. 1 ns)
was recovered that exhibits a negative amplitude at longer
wavelengths, which corresponds to the position of the 2.2 ns
By comparison of caged butyric acids 7 and 8, which
spectrum. Negative amplitudes for preexponentials (rise bear naphtho-oxazole units that differ in the ring fusion, a
times) are commonly associated with the formation of a linear naphtho[2,3-d]oxazole and an angular naphtho[1,2-
species in the excited state. To clearly identify the different d]oxazole, respectively, it was found that, although both
spectra, they were normalized, and in that of 12, the emis- cleaved readily at 254 and 300 nm, the irradiation time ob-
sion is completely dominated by the shorter-lived fluores- tained for the release of butyric acid from 8 was less than
cence (Table 2).
1 min.
Our research into the development of alternative photo-
Concerning the photolysis of 7 and 8 at 350 nm, the pho-
sensitive groups for the protection of bifunctional molecules tocleavage was not achieved in a practical period of time,
has involved the successful use of oxazole-based structures which was predicted from the values of wavelengths of
[
16]
for the first time.
coumarins) are well-established light-activated protecting irradiation time of 519 min (ca. 9 h) for 8.
groups, and we have been engaged in the application of Concerning 9 and 10, which bear angular coumarins 3-
In addition, 2-oxo-2H-benzopyrans maximum absorption in the solvent used with the lowest
(
their related fused derivatives, 3-oxo-3H-benzo[f]benzo- oxo-3H-benzo[f]benzopyran and 2-oxo-2H-benzo[h]benzo-
pyrans and 2-oxo-2H-benzobenzopyrans (benzocoumar- pyran, respectively, it was found that cleavage was faster at
[
12,14,17,18]
ins).
Both systems are suitable for photorelease all the wavelengths of irradiation for 10, especially at
purposes, their choice depends on the required wavelengths, 350 nm.
and have encouraged us to explore their use in other appli-
cations, namely, light-triggered drug delivery.
With this in mind and in order to achieve the best release
conditions of butyric acid, 11 and 12, which are based on
The evaluation of photoactive butyric acid prodrugs 7– the combination of 2-oxo-2H-benzopyran and oxazole in a
2, which possess naphtho[2,3-d]oxazole, naphtho[1,2-d]- fused system with an ester linkage to the active molecule
1
oxazole, 3-oxo-3H-benzo[f]benzopyran, 2-oxo-2H-benzo[h]- through a methylene spacer directly connected to the pyran
benzopyran and 6-oxo-6H-benzopyrano[6,7-d]oxazole, in or oxazole rings, respectively, were studied under irradiation
which the latter has the linkage between the heterocycle and under the same conditions as 7–10. At 254 and 300 nm, the
the active molecule through oxopyran or oxazole moieties, irradiation times of 11 and 12 were longer than for 7 and
was carried out by photolysis studies under irradiation at 8, whereas at 350 nm their photolytic behaviour was in-
–
4
different wavelengths. Solutions of 7–12 (1ϫ10 m) in ferior to that of 10, but superior to that of 7 and 8. Com-
methanol/HEPES buffer (80:20) solution were irradiated parison between the irradiation times of 11 and 12 revealed
with a Rayonet RPR-100 reactor at 254, 300, 350 and that cleavage of the ester linkage was improved for 11,
419 nm in order to determine the most favourable uncaging which bears butyric acid connected to the pyran ring, at
conditions. The course of the photocleavage reaction was all irradiation wavelengths tested. These data revealed the
monitored by reverse-phase-(RP)-HPLC with UV detec- contribution of both heterocyclic rings when combined in a
926
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 922–930

Photolytic Release of Butyric Acid from Heteroaromatic Cages
1
Figure 3. H NMR spectra in [D
4
]methanol/D
2
O (80:20) for the photolysis of 10 at 350 nm: (a) before irradiation, (b) after irradiation
for 60 min, (c) after irradiation for 140 min, (d) free butyric acid.
single system for the time required for the complete release sentative example). Variable irradiation times were required
of butyric acid from the ester cages. They are also consistent for the quantitative release of the butyric acid, which de-
with the time-resolved fluorescence data, which indicate pended on the structure of the heterocycle.
that the compounds that exhibit the more complex decay
kinetics have the fastest rate of photocleavage.
As butyric acid is an organic molecule of biological rele-
vance, cleavage and release could benefit from being carried
Conclusions
out by irradiation at longer wavelengths in practical times.
As a result, the behaviour of 9 and 10, which were the most 3H-benzo[f]benzopyran,
Naphtho[2,3-d]oxazole, naphtho[1,2-d]oxazole, 3-oxo-
2-oxo-2H-benzo[h]benzopyran
promising at 350 nm (263 min for 9 and 96 min for 10), and 6-oxo-6H-benzopyrano[6,7-d]oxazole (with a linkage
were also evaluated at 419 nm, which resulted in an ex- between the heterocycle and the active molecule through
pected increased, but still useful, irradiation time for 10 oxopyran or oxazole moieties) were used in the synthesis
(187 min) and an impractical result for 9 (3442 min, ca. of caged butyric acid through an ester linkage. Photolytic
5
7 h). In addition, the photolytic process at 300 nm was cleavage studies at 254, 300, 350 and 419 nm in methanol/
1
monitored by H NMR in a [D ]methanol/D O (80:20) HEPES buffer (80:20) solution revealed the possibility of
4
2
–
3
solution at a concentration of 9.0ϫ10 m (several times using these heterocyclic systems for the light-triggered re-
larger than that used in the experiments followed by lease of the active molecule in reasonable times. The use
HPLC), which led to an increase in the photolysis time for of time-resolved fluorescence techniques contributed to the
the complete release of the acid. During irradiation, the sig- elucidation of the dynamic behaviour of these systems.
nals related to the linked acid decreased gradually, with Overall, these results suggest that the heterocyclic cages
concomitant increase of its signals in the released form, as studied may be considered as promising alternatives as pho-
well as signals due to aromatic byproducts related to the toactive prodrugs for butyric acid as a model carboxylic
heterocyclic cage (see Figure 3 for 10 at 350 nm as a repre- acid drug.
Eur. J. Org. Chem. 2012, 922–930
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
927

M. S. T. Gonçalves et al.
FULL PAPER
CH
2
), 5.40 (s, 2 H, CH
2
Het), 7.46–7.53 (m, 2 H, 6-H and 7-H),
Experimental Section
7.91 (s, 1 H, 9-H), 7.95 (dd, J = 6.8 and 2.7 Hz, 1 H, 8-H), 7.99
General: All melting points were measured with a Stuart SMP3
melting point apparatus. TLC analyses were carried out with
13
(dd, J = 6.9 and 2.7 Hz, 1 H, 5-H), 8.18 (s, 1 H, 4-H) ppm.
C
NMR (100.6 MHz, CDCl
3
, 25 °C): δ = 13.55 (CH
Het), 106.66 (C-9), 117.89 (C-4), 124.80
C-7), 125.71 (C-6), 127.88 (C-8), 128.53 (C-5), 131.26 (C-8a),
31.70 (C-4a), 140.54 (C-3a), 149.50 (C-9a), 163.01 (C-2), 172.71
C=O) ppm. IR (KBr 1%): ν˜ = 2958, 2932, 2875, 1740, 1694, 1644,
3 2
), 18.28 (CH ),
0.25 mm thick precoated silica plates (Merck Fertigplatten Kiesel-
3
(
1
(
2 2
5.66 (CH ), 58.08 (CH
gel 60F254), and spots were visualised under UV light. Chromatog-
raphy on silica gel was carried out with Merck Kieselgel (230–
240 mesh). IR spectra were determined with a BOMEM MB 104
spectrophotometer. UV/Vis absorption spectra (200–700 nm) were
obtained with a Shimadzu UV/2501PC spectrophotometer. NMR
spectra were obtained with a Varian Unity Plus spectrometer at an
1
1
626, 1579, 1505, 1470, 1443, 1415, 1305, 1260, 1242, 1165, 1096,
–1
050, 1018, 956, 938, 900, 882, 867, 799, 767, 666 cm . HRMS
+
(
EI): calcd. for C16
H15NO
3
[M ] 269.1052; found 269.1058.
1
13
operating frequency of 300 MHz for H and 75.4 MHz for C or
a Bruker Avance III 400 instrument at an operating frequency of
(
Naphtho[1,2-d]oxazol-2-yl)methyl Butyrate (8): From the reaction
–
4
1
13
of 2 (0.109 g, 4.18ϫ10 mol) in DMF (4 mL), potassium fluoride
4
00 MHz for H and 100.6 MHz for C by using the solvent peak
as internal reference at 25 °C. All chemical shifts are given in ppm
by using δ (Me Si) = 0 ppm as a reference, and J values are given
–
3
(
0.073 g,
1.25ϫ10 mol)
and
butyric
acid
(0.037 g,
–
4
4.18ϫ10 mol), 8 was obtained as a brown oil (0.100 g, 89%). R
f
H
4
1
=
0.98 (ethyl acetate/n-hexane, 1:1). H NMR (400 MHz, CDCl
3
,
in Hz. Assignments were made by comparison of chemical shifts,
peak multiplicities and J values and were supported by spin de-
coupling-double resonance and two-dimensional heteronuclear cor-
relation techniques. Low- and high-resolution mass spectrometry
was performed at the C.A.C.T.I. – Unidad de Espectrometria de
Masas, at the University of Vigo, Spain. Commercially available
reagents were used as received.
2
2
7
1
5 °C): δ = 0.98 (t, J = 7.6 Hz, 3 H, CH
3
), 1.72 (sext, J = 7.6 Hz,
), 5.43 (s, 2 H, CH Het),
H, CH ), 2.43 (t, J = 7.6 Hz, 2 H, CH
2
2
2
.53 (dt, J = 6.8 and 0.8 Hz, 1 H, 6-H), 7.65 (dt, J = 6.8 and 0.8 Hz,
H, 5-H), 7.66 (d, J = 9.2 Hz, 1 H, 9-H), 7.79 (d, J = 9.2 Hz, 1
H, 7-H), 7.94 (d, J = 9.0 Hz, 1 H, 4-H), 8.46 (d, J = 7.6 Hz, 1 H,
8
1
1
3
-H) ppm. C NMR (100.6 MHz, CDCl
3 3
, 25 °C): δ = 13.51 (CH ),
8.25 (CH ), 35.67 (CH ), 58.07 (CH Het), 110.75 (C-9), 121.91
2
2
2
Synthesis of 2-(Bromomethyl)naphtho[1,2-d]oxazole 2: To a solution
of 1-aminonaphthalen-2-ol (0.300 g, 1.53ϫ10–3 mol) in polyp-
hosphoric acid (1.53 g) was added bromoacetic acid (0.320 g,
(
1
(
C-8), 125.42 (C-6), 126.41 (C-3a), 126.57 (C-7), 127.12 (C-5),
28.46 (C-4), 131.05 (C-3b), 136.09 (C-7a), 148.25 (C-9a), 159.55
C-2), 172.72 (C=O) ppm. IR (KBr 1%): ν˜ = 3067, 2966, 2936,
2
.30ϫ10–3 mol), and the mixture was stirred at 130 °C for 5 h. The
2
1
6
2
876, 1747, 1704, 1640, 1592, 1572, 1529, 1448, 1374, 1308, 1273,
259, 1164, 1101, 1046, 1029, 1005, 939, 880, 804, 749, 697,
65 cm . HRMS (EI): calcd. for C16H15NO [M ] 269.1052; found
3
69.1051.
reaction mixture was poured into iced water and stirred for 1 h to
give a fine grey precipitate. The solid was collected by filtration,
washed with cold water and dried in a vacuum oven. After purifica-
tion by chromatography using ethyl acetate/n-hexane (1:1) as elu-
–1
+
ent, 2 was obtained as a grey solid (0.400 g, 98%). M.p. 106.2–
(9-Methoxy-3-oxo-3H-benzo[f]benzopyran-1-yl)methyl Butyrate (9):
1
–4
1
(
07.4 °C. R
300 MHz, CDCl
and 1.2 Hz, 1 H, 6-H), 7.68 (dt, J = 6.9 and 1.2 Hz, 1 H, 5-H),
.70 (d, J = 6.6 Hz, 1 H, 9-H), 7.86 (d, J = 9.0 Hz, 1 H, 7-H), 7.98
f
= 0.97 (ethyl acetate/n-hexane, 1:1). H NMR
From the reaction of 3 (0.03 g, 1.09ϫ10 mol) in DMF (2 mL),
–
4
3 2
, 25 °C): δ = 4.73 (s, 2 H, CH
), 7.53 (dt, J = 6.9 potassium fluoride (0.019 g, 3.27ϫ10 mol) and butyric acid
–4
(0.011 g, 1.20ϫ10 mol), 9 was obtained as a brown solid (0.027 g,
80%). M.p. 123.7–124.9 °C. R = 0.58 (ethyl acetate/light petro-
leum, 3:7). H NMR (400 MHz, CDCl , 25 °C): δ = 1.01 (t, J =
7.6 Hz, 3 H, CH ), 1.74 (sext, J = 7.2 Hz, 2 H, CH ), 2.49 (t, J =
), 3.97 (s, 3 H, OCH ), 5.69 (d, J = 1.6 Hz, 2 H,
Het), 6.68 (d, J = 1.6 Hz, 1 H, 2-H), 7.21 (dd, J = 8.8 and
7
f
1
3
1
(d, J = 9.0 Hz, 1 H, 4-H), 8.48 (d, J = 6.6 Hz, 1 H, 8-H) ppm.
C
3
NMR (75.4 MHz, CDCl
3
, 25 °C): δ = 20.88 (CH
2
), 110.81 (C-9),
3
2
1
5
1
1
8
21.99 (C-8), 125.67 (C-6), 126.44 (C-3a), 127.12 (C-7), 127.39 (C- 7.6 Hz, 2 H, CH
2
3
), 128.62 (C-4), 131.21 (C-3b), 136.51 (C-7a), 148.62 (C-9a),
59.95 (C-2) ppm. IR (KBr 1%): ν˜ = 3100, 3055, 2974, 2924, 1700,
656, 1600, 1500, 1392, 1309, 1275, 1254, 1218, 1102, 1049, 926,
CH
2
2.4 Hz, 1 H, 8-H), 7.33 (d, J = 8.8 Hz, 1 H, 5-H), 7.48 (d, J =
2.4 Hz, 1 H, 10-H), 7.82 (d, J = 9.2 Hz, 1 H, 7-H), 7.91 (d, J =
–1
79
13
88, 813, 743, 687, 666 cm . HRMS (EI): calcd. for C12
H
8
81
NO Br
8.8 Hz, 1 H, 6-H) ppm. C NMR (100.6 MHz, CDCl
3
, 25 °C): δ
), 55.43 (OCH ), 64.01
Het), 105.73 (C-10), 111.98 (C-4b), 112.76 (C-2), 115.30 (C-
), 116.65 (C-8), 126.38 (C-6a), 130.64 (C-6b), 131.30 (C-7), 133.74
C-6), 151.17 (C-1), 155.54 (C-4a), 159.66 (C-9), 160.31 (C-3),
+
+
[M ] 260.9789; found 260.9778; calcd. for C12
H
8
NO Br [M ]
= 13.64 (CH
(CH
3
), 18.29 (CH
2
), 35.96 (CH
2
3
262.9769; found 262.9772.
2
5
(
Synthesis of 7–12
General Procedure for the Synthesis of 7–9, 11 and 12: To a solution
of the bromo- or chloromethyl precursor 1–3, 5 and 6 (1 equiv.) in
dry DMF (2 or 3 mL) were added potassium fluoride (3 equiv.) and
butyric acid (1 equiv.). The reaction mixture was stirred at room
temperature for 5 h or 2 d (9, 11 and 12). Potassium fluoride was
removed by filtration, the solvent was removed by rotary evapora-
tion under reduced pressure and the crude residue was purified by
column chromatography using mixtures of ethyl acetate and n-hex-
ane as eluent.
172.76 (C=O) ppm. IR (KBr 1%): ν˜ = 3416, 2964, 2931, 2252,
1718, 1625, 1595, 1518, 1461, 1444, 1425, 1406, 1363, 1335, 1299,
1277, 1231, 1198, 1105, 1060, 1023, 988, 949, 911, 846, 821, 732,
–
1
+
665, 646, 601 cm . HRMS (EI): calcd. for C19
326.1154; found 326.1171.
18 5
H O [M ]
(
(
(
2-Methyl-6-oxo-6H-benzopyrano[6,7-d]oxazol-8-yl)methyl Butyrate
–
4
11): From the reaction of 5 (0.029 g, 1.15ϫ10 mol) in DMF
–
4
2 mL), potassium fluoride (0.020 g, 3.45ϫ10 mol) and butyric
–
4
acid (0.010 g, 1.15ϫ10 mol), 11 was obtained as a yellow oily
1
(
Naphtho[2,3-d]oxazol-2-yl)methyl Butyrate (7): From the
solid (0.032 g, 95%). H NMR (300 MHz, CDCl
(t, J = 7.2 Hz, 3 H, CH ), 1.74 (sext, J = 7.2 Hz, 2 H, CH
(t, J = 7.2 Hz, 2 H, CH ), 2.7 (s, 3 H, CH Het), 5.36 (s, 2 H, CH
Het), 6.50 (s, 1 H, 7-H), 7.50 (s, 1 H, 4-H), 7.79 (s, 1 H, 9-H) ppm.
3
, 25 °C): δ = 1.00
–4
reaction of 1 (0.030 g, 1.15ϫ10 mol) in DMF (2 mL), potassium
fluoride (0.020 g, 3.45ϫ10 mol) and butyric acid (0.010 g,
1
M.p. 125.7–126.5 °C. R
NMR (400 MHz, CDCl
CH ), 1.75 (sext, J = 7.6 Hz, 2 H, CH
3
2
), 2.43
–4
2
3
2
.15ϫ10–4 mol), 7 was obtained as a yellow solid (0.030 g, 97%).
1
13
f
= 0.93 (ethyl acetate/n-hexane, 1:1).
H
C NMR (75.4 MHz, CDCl
), 35.88 (CH
112.13 (C-7), 113.44 (C-9), 114.36 (C-8a), 138.62 (C-9a), 149.55 (C-
3
, 25 °C): δ = 13.65 (CH
3 3
), 14.62 (CH
3
, 25 °C): δ = 1.01 (t, J = 7.6 Hz, 3 H, Het), 18.34 (CH
), 2.48 (t, J = 7.6 Hz, 2 H,
2
2
), 61.18 (CH Het), 99.58 (C-4),
2
3
2
928
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 922–930

Photolytic Release of Butyric Acid from Heteroaromatic Cages
8), 151.58 (C-4a), 152.67 (C-3a), 160.37 (C-6), 166.02 (C-2), 172.71
(by using DAS6 software) of the TRES measurements, which in-
(C=O) ppm. IR (KBr 1%): ν˜ = 3077, 3048, 3010, 2928, 1743, 1722, volved the collection of fluorescence decays for a fixed collection
1
1
7
3
642, 1606, 1578, 1477, 1437, 1430, 1394, 1382, 1354, 1283, 1273,
time over a selected wavelength range (at 5 nm intervals).
253, 1218, 1149, 1131, 1037, 1016, 962, 915, 901, 879, 869, 814,
–1
+
Photolysis General: Photolyses were carried out by using a Rayonet
RPR-100 chamber reactor equipped with 10 lamps of 254, 300, 350
and 419 nm. HPLC analyses were performed by using a Licrospher
100 RP18 (5 μm) column in a JASCO HPLC system composed by a
PU-2080 pump and a UV-2070 detector with ChromNav software.
34, 699, 681, 609 cm . HRMS (EI): calcd. for C16
01.0951; found 301.0953.
5
H15NO [M ]
(
(
(
8-Methyl-6-oxo-6H-benzopyrano[6,7-d]oxazol-2-yl)methyl Butyrate
12): From the reaction of 6 (0.031 g, 1.05ϫ10 mol) in DMF
2 mL), potassium fluoride (0.018 g, 3.15ϫ10 mol) and butyric
–4
–4
Photolysis Procedure: A 1ϫ10–4 m methanol/HEPES buffer (80:20)
solution of 7–12 (5 mL) was placed in a quartz tube and irradiated
in the reactor at the desired wavelength. HEPES buffer solution
was prepared in distilled water with HEPES (10 mm), sodium
chloride (120 mm), potassium chloride (3 mm), calcium chloride
–4
acid (0.010 g, 1.15ϫ10 mol), 12 was obtained as a white solid
0.015 g, 47%). M.p. 118.1–119.3 °C. R = 0.45 (ethyl acetate/light
petroleum, 3:7). H NMR (400 MHz, CDCl , 25 °C): δ = 1.00 (t,
J = 7.6 Hz, 3 H, CH ), 1.74 (sext, J = 7.2 Hz, 2 H, CH ), 2.48 (t,
J = 7.6 Hz, 2 H, CH ), 2.52 (d, J = 1.2 Hz, 3 H, CH Het), 5.37
s, 2 H, CH Het), 6.34 (d, J = 1.2 Hz, 1 H, 7-H), 7.52 (d, J =
.4 Hz, 1 H, 4-H), 7.96 (s, 1 H, 9-H) ppm. 13C NMR (100.6 MHz,
(
f
1
3
3
2
2
3
(
1 mm) and magnesium chloride (1 mm) and pH adjusted to 7.2.
(
0
2
Aliquots of 100 μL were taken at regular intervals and analysed by
RP-HPLC. The eluent was acetonitrile/water (3:1) at a flow rate of
CDCl
3
, 25 °C): δ = 13.58 (CH
Het), 99.64 (C-4), 114.27 (C-7), 115.63 (C-9),
17.89 (C-8a), 137.74 (C-9a), 152.0 (C-4a), 152.32 (C-8), 152.38 (C-
a), 160.40 (C-6), 162.52 (C-2), 172.68 (C=O) ppm. IR (KBr 1%):
3 2 3
), 18.29 (CH ), 19.21 (CH ), 35.65
0
.8 (7–9, 12) or 1.0 mL/min (10, 11) previously filtered through a
Millipore type HN 0.45 μm filter and degassed by ultrasound for
0 min. The chromatograms were traced by detecting UV absorp-
tion at the wavelength of maximum absorption for each conjugate
retention time: 7, 7.4; 8, 7.7; 9, 6.6; 10, 8.6; 11, 6.4; 12, 4.2 min).
(
1
3
2 2
CH ), 57.75 (CH
3
ν˜ = 3054, 2960, 2931, 2878, 1788, 1737, 1717, 1631, 1569, 1437,
(
1
1
6
3
404, 1385, 1368, 1341, 1316, 1280, 1241, 1210, 1167, 1130, 1053,
028, 950, 925, 908, 877, 858, 811, 782, 755, 742, 697, 665,
42 cm . HRMS (EI): calcd. for C16
01.0931.
Supporting Information (see footnote on the first page of this arti-
cle): Time-resolved emission spectra (TRES) for compounds 7–12
in methanol/HEPES buffer (80:20) solution.
–1
+
5
H15NO [M ] 301.0951; found
Synthesis of (6-Methoxy-2-oxo-2H-benzo[h]benzopyran-4-yl)methyl
Butyrate (10): To a solution of 4 (0.022 g, 8.83ϫ10–5 mol) in dry
Acknowledgments
–
4
DMF (2 mL) at 0 °C was added HOBt (0.024 g, 2.56ϫ10 mol)
followed by DCC (0.053 g, 2.57ϫ10–4 mol) after stirring for
Thanks are due to the Fundação para a Ciência e Tecnologia (Por-
tugal) for financial support through project PTDC/QUI/69607/
–4
1
0 min. After 10 min, butyric acid (1.6ϫ10–2 mL, 1.77ϫ10 mol)
was added and the reaction mixture was stirred at room tempera-
ture for 5 d. The solvent was evaporated and the crude residue dis-
solved in dichloromethane (10 mL). The organic phase was washed
with sodium carbonate (3ϫ10 mL) and dried with magnesium sul-
fate. Purification by chromatography in dichloromethane/methanol
2
006 (FCOMP-01-0124-FEDER-007449) and PhD grants to
A. M. P. (SFRH/BD/61459/2009) and A. M. S. S. (SFRH/BD/
0813/2011). The Bruker Avance III 400 NMR spectrometer is part
8
of the National NMR Network (RNRMN) and was purchased in
the framework of the National Program for Scientific Re-equip-
ment, contract REDE/1517/RMN/2005 with funds from POCI
(
1
100:1) gave 10 as a yellow solid (0.009 g, 30%). M.p. 124.6–
1
25.3 °C. R
f
= 0.88 (dichloromethane/methanol, 100:1). H NMR
, 25 °C): δ = 1.02 (t, J = 7.2 Hz, 3 H, CH ), 1.72–
.81 (m, 2 H, CH ), 2.48 (t, J = 7.2 Hz, 2 H, CH ), 4.04 (s, 3 H,
), 5.38 (d, J = 1.2 Hz, 2 H, CH Het), 6.59 (s, 1 H, 3-H),
2010 (FEDER) and FCT.
(400 MHz, CDCl
3
3
1
2
2
OCH
3
2
[1] V. J. Stella, R. T. Borchardt, M. J. Hageman, R. Oliyai, H.
Maag, J. W. Tilley in Prodrugs: Challenges and Rewards, part
1, vol. V, Springer, New York, 2007, pp. 703–729.
6
1
.65 (s, 1 H, 5-H), 7.65–7.70 (m, 2 H, 8-H and 9-H), 8.27–8.29, (m,
13
H, 7-H), 8.51–8.54 (m, 1 H, 10-H) ppm. C NMR (100.6 MHz,
, 25 °C): δ = 13.68 (CH ), 18.41 (CH ), 35.98 (CH ), 55.87
), 61.33 (CH Het), 95.27 (C-5), 112.22 (C-4a), 112.47 (C-
[
[
2] A. S. Kearney, Adv. Drug Delivery Rev. 1996, 19, 225–239.
3] B. Pavan, A. Dalpiaz, N. Ciliberti, C. Biondi, S. Manfredini,
S. Vertuani, Molecules 2008, 13, 1035–1065.
CDCl
3
3
2
2
(OCH
3
2
3
1
6
2
1
8
3
), 122.24 (C-7), 122.42 (C-10), 124.01 (C-10a), 127.36 (C-6a),
27.94 (C-9), 128.43 (C-8), 145.72 (C-10b), 149.86 (C-4), 152.30 (C-
), 160.80 (C-2), 172.84 (C=O) ppm. IR (KBr 1%): ν˜ = 2965, 2925,
853, 1749, 1727, 1613, 1595, 1564, 1505, 1472, 1455, 1422, 1384,
353, 1308, 1272, 1248, 1167, 1142, 1118, 1084, 1031, 990, 956,
[4] Z. Zhang, H. Hatta, T. Ito, S. Nishimoto, Org. Biomol. Chem.
2
005, 3, 592–596.
5] M. S. Kim, S. L. Diamond, Bioorg. Med. Chem. Lett. 2006, 16,
007–4010.
[
4
[6] G. Berkovitch, D. Doron, A. Nudelman, Z. Malik, A. Re-
phaeli, J. Med. Chem. 2008, 51, 7356–7369.
[7] P. R. Pouillart, Life Sci. 1998, 63, 1739–1760.
–1
+
58, 818, 778, 735, 666 cm . HRMS (EI): calcd. for C19
26.1154; found 326.1157.
18 5
H O [M ]
[
8] L. Spina, F. Cavallaro, N. I. Fardowza, P. Lagoussis, D. Bona,
C. Ciscato, A. Rigante, M. Vecchi, Digest. Liver Dis. Supp.
2007, 1, 7–11.
Time-Resolved Fluorescence Measurements: Time-resolved fluores-
cence measurements were performed with a HORIBA Scientific
FluoroCube-01 equipped with DeltaDiode excitation sources (run-
ning either at 8 or 10 MHz) and a TBX-07C detector. The exci-
tation wavelengths employed were 295 nm (LED source) for 7 and
[9] I. Levovich, A. Nudelman, G. Berkovitch, L. Swift, S. Cutts,
D. R. Phillips, A. Rephaeli, Cancer Chemother. Pharmacol.
2
008, 62, 471–482.
[
[
10] A. Brioschi, G. P. Zara, S. Calderoni, M. R. Gasco, A. Mauro,
Molecules 2008, 13, 230–254.
8
1
, 375 nm (laser source) for 9 and 10 and 317 nm (LED source) for
1 and 12. Data were analysed by using DAS6 software and the
11] U. Aich, M. A. Meledeo, S.-G. Sampathkumar, J. Fu, M. B.
Jones, C. A. Weier, S. Y. Chung, B. C. Tang, M. Yang, J. Hanes,
K. J. Yarema, Glycoconjugate J. 2010, 27, 445–459.
decays reconvoluted with the instrumental response and fitted to
the sum of exponentials. The goodness of fit was judged in terms
of a chi-squared value and weighted residuals. DAS, associating the
spectra with a decay time, were obtained from the global analysis
[12] A. M. Piloto, D. Rovira, S. P. G. Costa, M. S. T. Gonçalves,
Tetrahedron 2006, 62, 11955–11962.
Eur. J. Org. Chem. 2012, 922–930
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
929

M. S. T. Gonçalves et al.
FULL PAPER
[
13] A. S. C. Fonseca, M. S. T. Gonçalves, S. P. G. Costa, Tetrahe-
dron 2007, 63, 1353–1359.
14] M. J. G. Fernandes, M. S. T. Gonçalves, S. P. G. Costa, Tetra-
hedron 2008, 64, 3032–3038.
15] A. S. C. Fonseca, M. S. T. Gonçalves, S. P. G. Costa, Amino
Acids 2010, 39, 699–712.
16] A. M. S. Soares, S. P. G. Costa, M. S. T. Gonçalves, Tetrahe-
dron 2010, 66, 8189–8195.
[18] A. M. Piloto, A. M. S. Soares, S. P. G. Costa, M. S. T. Gon-
çalves, Amino Acids 2011, DOI: 10.1007/s00726-011-0969-0.
[
[
19] F. S. Tjoeng, G. A. Heavner, Synthesis 1981, 897–899.
[
20] J. V. Morris, M. A. Mahaney, J. R. Huber, J. Phys. Chem. 1976,
80, 969–974.
[
[
[
[
[
21] A. M. Piloto, S. P. G. Costa, M. S. T. Gonçalves, Tetrahedron
Lett. 2005, 46, 4757–4760.
22] R. Schmidt, D. Geissler, V. Hagen, J. Bendig, J. Phys. Chem. A
2007, 111, 5768–5774.
17] A. M. S. Soares, S. P. G. Costa, M. S. T. Gonçalves, Amino
Acids 2010, 39, 121–133.
Received: September 23, 2011
Published Online: December 8, 2011
930
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 922–930