Development of Green/Red-Absorbing Chromophores Based on a Coumarin Scaffold That Are Useful as Caging Groups

Article abstract of DOI:10.1021/acs.joc.7b00788

We report the design, synthesis, and spectroscopic characterization of a series of push-pull chromophores based on a novel coumarin scaffold in which the carbonyl of the lactone function of the original coumarin dyes has been replaced by the cyano(4-nitro

Full text of DOI:10.1021/acs.joc.7b00788

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Article
Development of green/red-absorbing chromophores
based on a coumarin scaffold useful as caging groups
Albert Gandioso, Sara Contreras, Ivanna Melnyk, Javier Oliva, Santi
Nonell, Dolores Velasco, Jaume Garcia-Amorós, and Vicente Marchán
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017
Downloaded from http://pubs.acs.org on May 5, 2017
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Development of green/red-absorbing chromophores based on a
coumarin scaffold useful as caging groups
†
†
†
†
§
Albert Gandioso, Sara Contreras, Ivanna Melnyk, Javier Oliva, Santi Nonell,
†
,‡
†
,†
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Dolores Velasco, Jaume GarcíaꢀAmorós, Vicente Marchán*
†
Secció de Química Orgànica, Departament de Química Inorgànica i Orgànica, IBUB
Universitat de Barcelona Martí i Franquès 1ꢀ11 Eꢀ08028 Barcelona (Spain)
Eꢀmail: vmarchan@ub.edu
‡
Institut de Nanociència i Nanotecnologia (IN2UB). Universitat de Barcelona. Eꢀ08028
Barcelona (Spain)
§
Institut Químic de Sarrià, Universitat Ramon Llull, Eꢀ08017 Barcelona (Spain)
TOC ABSTRACT GRAPHIC
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ABSTRACT
We report the design, synthesis and spectroscopic characterization of a series of pushꢀ
pull chromophores based on a novel coumarin scaffold in which the carbonyl of the
lactone function of the original coumarin dyes has been replaced by the cyano(4ꢀ
nitrophenyl)methylene moiety. The skeleton of the compounds was synthesized by
condensation of a thiocoumarin precursor with the corresponding arylacetonitrile
derivatives, and their photophysical properties were fineꢀtuned through the
incorporation of electronꢀwithdrawing groups (EWG) like nitro and cyano at the phenyl
ring, leading to absorption in the green to red region. Although fluorescence emission
was weakened or even cancelled upon introduction of two or three strong EWGs, it is
noticeable the emission of the mononitroꢀcontaining coumarin derivatives in the red
region upon excitation with green light, as well as their significant large Stokes’ shifts.
The new coumarin derivatives can be useful as photocleavable protecting groups, as
demonstrated through the synthesis and characterization of a series of coumarinꢀbased
photocages of benzoic acid. Preliminary photolysis studies with green light have
demonstrated that the structure of the coumarin chromophore influences on the rate of
the uncaging process, opening the way to exploiting these new coumarin scaffolds as
caging groups removable with visible light.
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INTRODUCTION
The use of photocleavable protecting groups (PPGs or caging groups) has received wide
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attention in recent years to regulate the activity of biologically relevant molecules,
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including drugs based on small compounds and larger biomolecules (peptides and
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proteins and oligonucleotides). The reason relies on the possibility of controlling the
release of active species from the corresponding nonꢀbiologically active precursors
(caged compounds) by using light as an external trigger. Besides offering high
spatiotemporal precision, light does not contaminate the living system and its
wavelength and intensity can be precisely regulated to make it fully compatible with
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living systems. PPGs have also demonstrated great potential in many other fields,
1b
including organic synthesis, materials science, industry and agriculture.
Taking into account the potential of caged compounds as therapeutic agents and as
optical tools to study and control complex chemical and biological processes, the
selection of the appropriate PPG is of great importance. An ideal caging group must
fulfill several requirements including synthetic feasibility and stability under
physiological conditions, together with a compatibility with a variety of leaving groups.
In addition, it should have a large molar extinction coefficient to allow an efficient
uncaging and should not generate toxic byꢀproducts. The majority of photoremovable
protecting groups reported to date require UV irradiation for photoactivation, which
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should be avoided given its known phototoxicity and capacity to interact with
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endogenous biomolecules. Moreover, the low capacity of penetration of UV light (even
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in the UVA range) into deep tissues compromise further therapeutic applications. In
recent years, some promising achievements with PPGs removable upon oneꢀphoton
1bc
excitation in the 400ꢀ500 nm range have been described. However, further work is
necessary for the development of new longꢀwavelength absorbing caging groups
removable with oneꢀphoton red light (>600 nm). This nonꢀdamaging radiation offers
higher efficiencies than alternatives based on twoꢀphoton processes and ensures deeper
penetration in mammalian tissues due to reduced absorbance. In addition, the
availability of redꢀlight absorbing photocages would eventually facilitate the design of
complex wavelengthꢀselective uncaging systems that could sequentially remove two or
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more PPGs from a single compound by using different wavelengths of light.
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With this idea in mind, we decided to choose the N,Nꢀdiethylamino(coumarin 4ꢀ
yl)methyl moiety as a starting point since the diethylamino electronꢀdonating group
(EDG) at position 7 causes larger red shifts in the absorption spectrum than other EDG
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substituents such as alkoxy. Moreover, polar groups such as carboxylates could be
eventually appended to the aniline moiety to increase, if necessary, the aqueous
solubility of the caged compound without significantly compromise the spectral
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properties of the chromophore. Recent studies have also demonstrated that
modification of N,Nꢀdialkyamino substituents at the coumarin’s 7ꢀposition can be used
to improve the photophysical properties of fluorescent dyes such as brightness,
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photostability and quantum yield. This coumarinylmethyl platform is easy to
synthesize and amenable to structural modifications allowing the attachment of the
molecule to be caged through a variety of bonds, including esters, amides, carbamates
1bc
or carbonates. In recent years, thionation of the carbonyl group of the lactone has
been used to shift the absorption maximum, which allowed to trigger uncaging with
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blueꢀlight in different systems, as reported by Jullien,
and Costa groups.
Interestingly, the incorporation of two cyano groups at position 2 led to
dicyanomethylenecoumarinylmethyl derivatives with maximum absorption around 500
8,13
nm in the 7ꢀdiethylamino series.
This modification has also been incorporated in
coumarinꢀcaged cyclic morpholino oligonucleotides by Deiters, Chen and collaborators
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for sequential gene silencing, as well as by us in a caged cyclic RGD peptide for
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applications in photocontrolled targeted therapies.
Based on these precedents, in this work we have focused on the design and synthesis of
new coumarinꢀbased chromophores and explored their applications as visible lightꢀ
removable PPGs. As shown in Scheme 1, we reasoned that replacement of one cyano
group in the dicyanomethylenecoumarin by a phenyl ring containing electronꢀ
withdrawing groups (EWG) at ortho and para positions would increase the pushꢀpull
15
character of the chromophore; this would result in a large bathochromic shift in the
absorption compared with previously reported coumarin derivatives, a typical
characteristic of the intramolecular chargeꢀtransfer effect. In the first section of this
report we describe the design, synthesis and spectroscopic characterization of a small
library of model coumarinylmethyl derivatives that explore the effect of EWGs on the
photophysical properties of the new chromophores (Scheme 1). In the second section,
the best candidates have been functionalized at position 4 of the coumarin moiety
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through an ester bond and their suitability as photocages of carboxylic acids has been
explored, using benzoic acid as a model. The photophysical and photochemical
properties of two caged model compounds, particularly their photoactivation upon oneꢀ
photon excitation with green light have also been investigated.
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O
Ph
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O
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CN
CN
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Et N
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Et N
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O
Et N
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O
R
2
Et N
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O
R
R'
R"
R"
R, R', R" = H and/or EWG (F, CN, NO2)
X= H or CH3
Scheme 1. Design of new coumarinꢀbased chromophores and caging groups by replacement of
a cyano group in dicyanocoumarin by a phenyl ring containing EWG at ortho and para
positions.
RESULTS AND DISCUSSION
Design and synthesis of new coumarin-based chromophores with red-shifted
absorption
As shown in Scheme 2, we have synthesized a small library of 2ꢀ(cyano(4ꢀ
nitrophenyl)methylene coumarin derivatives (4-9) in which one cyano group of the
parent dicyanocoumarin (3) has been replaced by a 4ꢀnitrophenyl substituent containing
one or two substituents (e.g. cyano, nitro or fluoro) at the ortho positions of the
aromatic ring. Since at this stage we wanted to assess the influence of that substitution
on the absorption and emission properties of new chromophores, we kept the methyl
group at position 4 unaltered. First, 7ꢀ(N,Nꢀdiethylamino)ꢀ4ꢀmethylꢀ2ꢀthiocoumarin (2)
16
was synthesized in 92% yield by reaction of 1 with Lawesson’s reagent in toluene at
reflux overnight. Condensation of such thionated coumarin with malononitrile in the
3
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presence of triethylamine and silver nitrate
afforded the parent
dicyanomethylenecoumarin 3 in 86% yield after column chromatography.
Next, the reaction of thiocoumarin 2 with the commercially available 4ꢀ
nitrophenylacetonitrile (10) led to the desired coumarin derivative 4 (Scheme 2). No
reaction occurred with phenylacetonitrile, whose acidity is much lower than that of 10
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(
a a
pK =12.3) or malononitrile (pK =11.1) . Taking into account these results and the
spectroscopic properties of 4 (see below), we selected the 4ꢀnitrophenyl moiety for
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exploring the incorporation of different substituents at the ortho positions of the
2
aromatic ring (CN, F and NO ) with the aim of tuning the photophysical properties of
the coumarin chromophore. All the required arylacetonitrile compounds (11-15) were
synthesized, except 2ꢀ(2,4,6ꢀtrinitrophenyl)acetonitrile (13), which was commercially
available.
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Scheme 2. Synthesis of coumarinꢀbased chromophores 3-9. a) Lawesson’s reagent, toluene,
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00ºC, 12 h, 92%; b) Malononitrile or arylacetonitrile derivatives 10-15, triethylamine, silver
nitrate, anhydrous ACN, rt, 2ꢀ4 h, 86% (3), 40% (4), 54% (5), 68% (6), 27% (7), 42% (8), 55%
9).
(
As shown in Scheme 3, 2ꢀ(2ꢀcyanoꢀ4ꢀnitrophenyl)acetonitrile (11) was obtained from 2ꢀ
cyanophenylacetonitrile by nitration with concentrated nitric acid and sulfuric acid at
low temperature in 60% yield. 2ꢀ(2,4ꢀdinitrophenyl)acetonitrile (12) was synthesized in
two steps from 2,4ꢀdinitrotoluene. First, reaction with N,Nꢀdimethylformamide dimethyl
acetal provided the corresponding enamine intermediate (16), which was reacted with
hydroxylamine hydrochloride and formic acid for 5 h at 110ºC to give 12 (60% yield)
after purification by silica column chromatography. The fluorinated arylacetonitrile
derivatives, 2ꢀ(2ꢀfluoroꢀ4ꢀnitrophenyl)acetonitrile (14) and 2ꢀ(2,6ꢀdifluoroꢀ4ꢀ
nitrophenyl)acetonitrile (15) were synthesized in two steps from 1,2ꢀdifluoroꢀ4ꢀ
nitrobenzene and 1,2,3ꢀtrifluoroꢀ4ꢀnitrobenzene, respectively. Reaction with ethyl
cyanoacetate and K CO in the presence of a catalytic amount of KI provided the
2
3
corresponding alkylated intermediates (17 and 18). After hydrolysis of the ester and
decarboxylation by reaction with HCl in a H O/AcOH mixture, the desired
2
arylacetonitrile derivatives were obtained in moderate yields 14 (32%) and 15 (39%).
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3 2 4
Scheme 3. Synthesis of arylacetonitrile derivatives 11-12 and 14-15. a) HNO , H SO , ꢀ5ºC, 3
h, 60% (11); b) N,Nꢀdimethylformamide dimethylacetal, anhydrous DMF, heat at reflux (16), 15
h, Ar atmosphere, 96% (16); c) hydroxylamine hydrochloride, formic acid, heat at reflux, 5 h,
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2 3
0% (12); d) ethyl cyanoacetate, K CO , KI, anhydrous DMF, 16 h at rt and 2 h at 100ºC, 98%
(
17), 72% (18); e) HCl, AcOH, 12 h, 100ºC, 32% (14), 39% (15).
Coumarins 5-9 (Scheme 2) were synthesized in a similar way that compound 4 by
reacting 2 with the corresponding arylacetonitrile derivatives (11-15). All the
compounds were isolated by silica column chromatography with moderate to good
yields and their purity was assessed by reversedꢀphase HPLC analysis, revealing a
single peak in all cases (Figure S4). Characterization was carried out by usual
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spectroscopic techniques such as high resolution ESI mass spectrometry and H, C and
1
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1
F NMR. It is worth noting that the H NMR spectra of most of the compounds
(except for 6 and 7) showed the presence of two sets of proton signals in CDCl and in
DMSOꢀd at 25ºC in an ∼ 95:5 (4; see Figure 1), 90:10 (5) or 80:20 ratio (8 and 9). The
3
6
13
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same duplicity was found in the C NMR spectra as well as in the F NMR spectra of
the fluorinated compounds. Taking into account the strong pushꢀpull character of the
coumarin derivatives, the exocyclic C=C bond connecting the Cꢀ2 of the coumarin
moiety and the Cꢀ4 of the pꢀnitrophenyl cannot be considered a pure double bond but a
double bond with partial single bond character due to πꢀdelocalization. Hence, we
hypothesized that rotation around this bond could afford a mixture of two rotamers
slowly interconverting in solution rather than two isolated diastereomers. The presence
of chemical exchange crossꢀpeaks between the resonances of the two species in the 2D
NOESY spectra of compound 4 (Figure 1) as well as in 2D EXSY experiment
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confirmed the presence of rotamers around the exocyclic C=C bond. The exchange rate
ꢀ1
for the rotameric equilibrium (kex = 0.13 s ) was estimated from the intensities of the
exchange crossꢀpeaks and is in agreement with a slow equilibrium on the chemical shift
1
time scale. Full assignment of the H NMR spectra of 4 with COSY and NOESY
experiments (Figures 1, S1ꢀS3) allowed to conclude that the E-rotamer was the one
preferred in all cases (as drawn in Schemes 1 and 2). The fact that the chemical shift of
the proton at position 3 of the coumarin moiety appears at slightly higher δ in the major
isomer (e.g. 6.71 ppm in 4) compared with the minor isomer (e.g. 6.64 ppm in 4) can be
attributed to the shielding effect of the phenyl ring over proton H3 in the Z
configuration. A similar trend was found for the chemical shift of the methyl group at
position 4 in both isomers (e.g. 2.40 ppm in the Eꢀrotamer vs 2.28 ppm in the Zꢀrotamer
of 4). As shown in Figure 1 and Figures S1, diagnostic NOE crossꢀpeaks such as the
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one between H3 proton of the coumarin in the Z isomer and H protons of the pꢀ
m
nitrophenyl ring provided further evidence of the structure of the two interconverting
rotamers.
NOE
A
H3: 6.64 ppm
Hm: 7.69 ppm
H3: 6.71 ppm
H3
CH3
CH3
H3
Hm
CN DMSO-d6
Et2N
O
Et2N
O
NO2
H8 Hm
NOE
NC
H8: 6.74 ppm
Hm: 8.03 ppm
NO2
E-rotamer
Z-rotamer
B
Hm E
H5 E
Hm Z
H3 E
H8 E
H6 E
Ho E
H5 Z
H8 Z
H6 Z
H3 Z
Ho Z
Exchange peak between
H8 E and H8 Z
Exchange peak between
Hm E and Hm Z
NOE
H3-Hm
Z rotamer
NOE
H8-Hm
E rotamer
Figure 1. A) Structure of E and Z rotamers of compound 4 with some diagnostic NOE crossꢀ
m
peaks indicated. B) Expansion of the 2D NOESY spectrum (t = 500 ms, 25ºC) of compound 4
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in DMSOꢀd
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showing exchange crossꢀpeaks between rotamer resonances of the same sign as the
diagonal.
Optical and photophysical properties of coumarin-based chromophores
The optical and photophysical properties (absorption and emission spectra, molar
absorption coefficients and fluorescence quantum yields) of the new coumarin
chromophores (4-9) were investigated in two solvents of different polarity (DCM and
MeOH). The UVꢀvis absorption spectra of the compounds are shown in Figures 2 and
Figures S6ꢀS7 of the Supporting Information, and their optical and photophysical
properties are summarized in Table 1 and Table S1 together with those of the parent
dicyanocoumarin (3) which was included for comparison purposes. First, it is worth
noting that replacement of one cyano group in 3 by pꢀnitrophenyl ring (compound 4)
was positive since it caused a 22ꢀnm bathochromic shift in DCM, thus indicating that
this modification increases the pushꢀpull character from the electronꢀdonating group
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(
NEt ) to the electronꢀwithdrawing group (NO ). As expected, this shift to longer
2
2
wavelengths was further increased when a second electronꢀwithdrawing substituent like
cyano (5) or nitro (6) where introduced in one of the two ortho positions, leading to
absorption maxima of 519 nm (Table S1) and 558 nm, respectively. However, the
introduction of one (8) or two (9) fluoro substituents was not beneficial since the
absorption maximum shifted slightly to shorter wavelengths (to 485 nm and 476 nm,
respectively; Table S1) when compared with 4, which can be attributed to the
mesomeric electronꢀdonating character of this substituent. The case of the trinitro
coumarin derivative (7) is particularly appealing since the absorption maximum was
shifted to 607 nm, being the absorbance still quite significant in the farꢀred region.
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,0
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,6
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Wavelength (nm)
Wavelength (nm)
Figure 2. Comparison of the UVꢀvis spectra of selected coumarin derivatives 3, 4, 6 and 7 (left)
and of fluorescence emission spectra of 3 and 4 (right) in DCM at RT.
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3
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4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
The molar absorption coefficients were also strongly influenced both by the nature and
ꢀ1
ꢀ1
ꢀ1
the number of the EWG at the phenyl ring (e.g. ε(4) = 31 mM cm vs ε(7) = 14 mM
ꢀ1
cm ). As shown in Figure 3, all the new coumarin derivatives led to red to blue
coloured solutions to the naked eye. It is also worth noting that all the coumarin
derivatives (4-9) showed an additional absorption band in the range 365ꢀ400 nm, whose
maximum absorption was also dependent on the nature and number of EWGs (Table
S1). As shown in Table 1, the maximum absorption of 4 was slightly blueꢀshifted in
MeOH (6 nm) with respect DCM, which is the same behaviour than of the parent
coumarin 3 (blueꢀshifted by 2 nm). The dependence of the absorption and emission
characteristics of 7ꢀdialkylaminocoumarins with solvent polarity is usually attributed to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
19
the strong intramolecular charge transfer character (ICT) of their excited states. Such
negative solvatochromism was particularly dramatic in the case of the dinitro and
trinitroꢀcontaining compounds (Table 1). For example, the maximum absorption of 7
was shifted from 607 nm (DCM) to 567 nm (MeOH) with increasing solvent polarity.
Figure 3. Photographic images of coumarin derivatives 3ꢀ9 in DCM under day light.
Table 1. Photophysical properties of coumarin derivatives 3, 4, 6 and 7.
Absorption
Emission
Stokes’shift
ε(λ
)
e
max
(mM cm )
a
-1
-1
b
c
d
φ
F
λ
max [nm]
λ
max [nm]
(nm)
Comp.
MeOH
DCM
MeOH
38
DCM
38
MeOH
519
DCM
510
MeOH
42
DCM
31
MeOH
0.05
DCM
0.02
3
4
477
479
360 / 493
379 / 539
396 / 567
366 / 499
385 / 558
402 / 607
19 / 30 20 / 31
19 / 18 22 / 19
15 / 10 20 / 14
408 / 598
414 / 640
105
141
<0.001
<0.001
f
_
_
_
_
_
_
6
436 /
415 /
f
_
_
_
_
_
_
7
449 /
418 /
a
b
Wavelength of the absorption maximum. Molar absorption coefficients at both λmax indicated.
Wavelength of the emission maximum upon excitation at a λEx positioned within each absorption band.
c
d
e
Stokes’ shift for the bands located in the visible region. Fluorescence quantum yield determined at 25ºC
F
using either 7ꢀdiethylaminoꢀ4ꢀmethylꢀcoumarin (coumarinꢀ1, λEx = 360 nm, ϕ = 0.73 in ethanol) or
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f
F
fluorescein (λEx = 460 nm, ϕ = 0.92 in aqueous 0.1 M NaOH) as a standard. No fluorescence detected
upon excitation at a λEx within the band located in the visible region.
Interestingly, coumarin 4 exhibited very weak dual luminescence in DCM with two
maximum emission bands centered in the blue (414 nm) and far red (640 nm) regions.
However, the overlapping of the emission bands prevented an accurate determination of
the fluorescence quantum yield. Nevertheless, it is worth noting the large Stokes’shift of
this compound compared with the parent dicyanocoumarin (141 nm for 4 vs 31 nm for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
, Table 1). Although the emission maximum was also blueꢀshifted when solvent
polarity was increased (from 640 nm in DCM to 598 nm in MeOH), the Stokes’ shift
was still considerably high for a 7ꢀdialkylaminocoumarin derivative in a polar solvent
(
104 nm). Fluorophores exhibiting large Stokes’ shifts are particularly interesting for
Försterꢀtype resonance energy transfer (FRET) applications since excitation and
emission bands are completely separated. The loss of fluorescence emission in
coumarin 4 compared to 3 is most likely the result of enhanced intramolecular electronꢀ
transfer quenching by the nitrophenyl moiety, leading to the formation of a weakly
emitting twisted ICT (TICT) excited state (typically nonꢀemissive in 7ꢀ
10
dialkylaminocoumarins and related aminoꢀcontaining dyes). Indeed, examples of the
quenching of the native fluorescence of organic dyes through incorporation of nitro
20
substituents are found in the literature. As expected, dinitro and trinitroꢀcontaining
coumarins (6 and 7) only showed an extremely weak fluorescence upon excitation at the
maximum absorption of the band located in the violetꢀblue region (Table S1) but no
emission was detected upon excitation with visible light.
Photocaging of benzoic acid with nitro-containing coumarin-based derivatives:
synthesis and photochemical properties
The overall results indicate that replacement of one cyano group in coumarin 3 by a
phenyl ring containing strong EWGs (e.g. NO
chromophores with attractive absorptivity in the biologically benign wavelength range
500ꢀ600 nm). Unfortunately, the weak fluorescence of the new coumarin derivatives
2
and CN) led to coumarinꢀbased
(
upon excitation in the visible region rules out their use as fluorophores, particularly the
diꢀ and trinitroꢀcontaining molecules. For this reason, we decided to explore alternative
application such as their use as caging groups removable with visible light by
functionalizing the methyl group at position 4 of the coumarin skeleton with an alcohol.
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
This approach has been widely exploited to cage several functionalities such as
1bc
carboxylic acids, amines, thiols or alcohols by using the appropriate chemical bond.
As shown in Scheme 4, we selected benzoic acid as a model compound to be caged with
nitro, dinitro and trinitroꢀcontaining coumarinylmethyl derivatives through the
formation of an ester bond (25, 26 and 27, respectively). The synthesis of the caged
model compounds was planned from 4ꢀ(acetoxymethyl)ꢀ7ꢀ(N,Nꢀdiethylamino)ꢀ2ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
thiocoumarin (22), a key intermediate described by Jullien and collaborators that can
be easily prepared from the commercially available 1 in four steps. Hydrolysis of the
acetate with HCl followed by esterification with benzoic acid afforded thiocoumarin 24,
which was reacted with the corresponding arylacetonitrile derivatives (10, 12 and 13) to
afford the expected caged model compounds (25-27) as single spots on TLC after silica
column chromatography. The three compounds were fully characterized by HR ESIꢀMS
1
13
and H and C NMR. As previously found with compound 4, two sets of proton signals
in an 90:10 ratio were identified in the NMR spectra of 25, whereas a single set of
∼
signals was observed for dinitro and trinitro coumarin derivatives. Interestingly, in this
case reversedꢀphase HPLC analysis of 25 showed two peaks (R = 26.1 and 27.9 min,
t
see Figure S5) in an
∼ 90:10 ratio. After isolation of the peaks, HPLC reꢀanalysis led to
the same initial mixture in both cases, which confirmed the presence of two rotamers
under equilibrium and reproduced the behaviour of the model coumarin 4.
Recently, we have demonstrated that both the structure of the coumarin chromophore
and the nature of the leaving group have a strong influence on the rate and efficiency of
21
the uncaging process. Indeed, the incorporation of a methyl group at the 4ꢀposition of
dicyanocoumarinylmethyl (DEAdcCM) derivatives led to new dicyanocoumarinylethyl
(DEAdcCE)ꢀphotocleavable protecting groups that can be removed upon green light
irradiation more efficiently from the corresponding caged carboxylic acids and
21
amines. This result can be attributed to the higher stability of the secondary
carbocation intermediate generated upon photoheterolysis of the CꢀO bond in the
corresponding caged compounds. Taking into account these precedents, we decided to
synthesize the caged model compound 28 (Scheme 4) with the aim of comparing its
photolysis with that of the nonꢀmethylated analogue (25). Compound 28 was efficiently
21
synthesized by reaction of the thiocoumarin precursor 32 (Scheme 4) with
arylacetonitrile 10. According to HPLC and NMR analysis, two rotamers in an 85:15
∼
ratio were also identified for compound 32, which parallels the behavior of 4 and 25.
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25
26
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. Synthesis of caged model compounds 25-28. a) SeO , dioxane/H O 1:0.03 (v/v), heat
2
2
4
at reflux, 15 days, 40%; b) NaBH , EtOH, 4 h, RT, 90%; c) AcOH, EDC·HCl, DMAP, DCM,
12 h, RT, Ar, 73%; d) Lawesson’s reagent, toluene, 15 h, heat at reflux, 76%; e) HCl, EtOH, 20
h, 90ºC; f) Benzoic acid, EDC·HCl, DMAP, DCM, 18 h, RT, Ar, 72% (2 steps e + f); g) first,
arylacetonitrile derivatives 10, 12 or 13 + NEt in anhydrous ACN or ACN/DCM 1:1 2 h RT,
3
3 3
then AgNO 2 h RT, 70% (25), 41% (26), 8% (27); h) CH MgCl, anh. THF, 4 h, ꢀ78ºC, 71%; i)
Benzoic acid, EDC·HCl, DMAP, DCM, 18 h, RT, Ar; j) Lawesson’s reagent, toluene, 15 h, heat
3
at reflux, 69% (2 steps i + j); k) first, 10 + NEt in anhydrous ACN/DCM 1:1 2 h RT, then
AgNO 2 h RT, 69% (28).
3
By comparison of the photophysical data (Figure 4 and Table 2) of the three caged
derivatives (25-27) with those of the corresponding model compounds (4, 6 and 7,
respectively; see Figure 2 and Table 1), the same tendency was found: the absorption
maxima shifted to increasingly longer wavelengths upon incorporation of one, two or
three nitro substituents at the ortho and para positions of the phenyl ring conjugated at
the 2ꢀposition of the coumarin moiety. However, replacement of the methyl group at
position 4 by benzoyloxymethyl caused a slight blueꢀshift in the absorption maximum
of the dinitroꢀ (558 nm in 6 vs 551 nm in 26) and the trinitroꢀ (607 nm in 7 vs 596 nm in
27) coumarin derivatives. Similarly, the molar absorption coefficients were strongly
influenced by the nature and the number of the EWG at the phenyl ring, and only the
mononitroꢀcontaining compound showed weak fluorescence upon excitation in the
visible region (Table 2). In agreement with our previous results with dicyanocoumarin
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
6
2
1
derivatives, the incorporation of the methyl group at the coumarin skeleton caused an
additional redꢀshift when compared with nonꢀmethylated analogues (e.g. compare 25
and 28) as well as a slight increase of the molar absorption coefficient.
35
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
30
2
5
6
2
25
20
15
10
5
0
27
28
300
400
500
600
700
800
Wavelength (nm)
Figure 4. Comparison of the UVꢀvis spectra of coumarin derivatives 25-28 in DCM at RT.
Table 2. Photophysical properties of coumarin derivatives 25-28 in DCM.
Absorption
Emission
Stokes’shift
ε(λ1max
)
-1 b
Comp.
a
c
-
d
λ
max [nm]
(mM cm )
λ
max [nm]
(nm)
2
5
366 / 501
386 / 551
405 / 596
367 / 503
18 / 25
414 / 634
133
e
_
_
26
18 / 16
16 / 11
441 /
e
_
_
27
445 /
28
22 / 32
414 / 635
132
a
c
b
Wavelength of the two absorption maximum. Molar absorption coefficients at both λmax indicated.
Wavelength of the emission maximum upon excitation at a λEx positioned within each absorption band.
Stokes’ shift for the bands located in the visible region. No fluorescence detected upon excitation at a
Ex located within the band in the visible region.
d
e
λ
Preliminary photolysis studies were carried out by irradiating a solution of caged model
3
f
compounds 25 and 28 in MeOH at 37ºC with a 505 nm LED source, and the course of
the uncaging process was monitored by reversedꢀphase HPLCꢀESI MS. As shown in
Scheme 5 and Figures S8ꢀS9, photoactivation of 25 and 28 yielded the corresponding
photolytic byꢀproducts 33 and 34, respectively, as well as the uncaged benzoic acid. The
formation of methoxyꢀcoumarin derivatives supports a solventꢀassisted photoheterolytic
bond cleavage upon electronic excitation via a cationic intermediate that reacts with
methanol. It is worth noting that the photorelease of the caged model compounds was
very slow under the conditions employed, particularly in the case of coumarin 25.
Indeed, only a 2% of deprotection was achieved after 5 h of irradiation, and this
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percentage was only increased to 4 % after 10 h. By contrast, photolysis of coumarin 28
was considerably more efficient (11% and 18% of deprotection after 5 h and 10 h of
irradiation, respectively), which can be attributed to the different stability of the
carbocation intermediates (primary in 25 and secondary in 28) generated during
photoheterolysis of the ester. This is in good agreement with our previous results with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
1
dicyanocoumarinꢀcaged model compounds. Finally, the uncaging quantum yields
(
(
φPhot) of the two caged model compounds were evaluated upon irradiation at 505 nm
21
ꢀ7
see the Experimental Section for further details) as
φ
Phot = 5.3 x 10 for 25 and
φ
Phot
=
ꢀ6
2
.3 x 10 for 28, which agrees with the results from the photolysis studies.
X
O
Ph
X
X
OMe
O
O
O
h
MeOH
CN
CN
+
CN
Et N
O
Et N
O
Et N
2
O
2
2
Ph
O
+
Ph
OH
MeOH
37ºC
X = H
X = CH3 28
25
X = H
X = CH3 34
33
NO2
NO2
NO2
Scheme 5. Photolysis of coumarin derivatives 25 and 28 with green light.
CONCLUSIONS
In summary, we have designed and synthesized a series of pushꢀpull chromophores
based on a novel coumarin scaffold containing a cyano(4ꢀnitrophenyl)methylene moiety
at position 2. The compounds can be readily prepared through condensation of a
thiocoumarin precursor with arylacetonitrile derivatives in the presence of triethylamine
and silver nitrate. The optical and photophysical properties of the chromophores can be
fineꢀtuned by incorporating electronꢀwithdrawing groups such as nitro and cyano at the
ortho and para positions of the phenyl ring, which shifts the absorption bands to the
visible region of the electromagnetic spectrum (500ꢀ650 nm) in a solventꢀdependent
manner. Notably, green light excitation of the mononitroꢀcontaining coumarins led to
emission in the red region with significant large Stokes’shifts even in polar solvents,
although with very low quantum yields. Unfortunately, the incorporation of two or three
strong EWGs weakened or even cancelled the fluorescence emission of the compounds,
which agrees with previous findings on other nitroꢀcontaining dyes. On the other hand,
the compounds were further used to synthesize a series of coumarinꢀbased photocages
of benzoic acid to ascertain their use as photocleavable protecting groups removable
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with visible light. Preliminary photolysis studies with green light have demonstrated
that the structure of the coumarin chromophore has a strong influence on the rate of the
uncaging process. Work is in progress to increase the redꢀshifted properties of the new
coumarin derivatives described in this work to facilitate uncaging in the farꢀred and
2
4
nearꢀIR region, as well as to improve their photophysical properties, especially
fluorescence emission.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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3
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5
6
7
8
9
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3
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8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
Materials and Methods
Unless otherwise stated, common chemicals and solvents (HPLC grade or reagent grade
quality) were purchased from commercial sources and used without further purification.
2ꢀ(2,4,6ꢀtrinitrophenyl)acetonitrile (13) was purchased from SelectLab Chemicals
GmbH. Aluminium plates coated with a 0.2 mm thick layer of silica gel 60 F254 were
used for thinꢀlayer chromatography analyses (TLC), whereas flash column
chromatography purification was carried out using silica gel 60 (230ꢀ400 mesh).
Reversedꢀphase highꢀperformance liquid chromatography (HPLC) analyses were
carried out on a Jupiter Proteo C18 column (250x4.6 mm, 90 Å 4
mL/min) using linear gradients of 0.045% TFA or 0.1% formic acid in H
.036% TFA or 0.1% formic acid in ACN (B). NMR spectra were recorded at 25ºC in a
ꢀm, flow rate: 1
2
O (A) and
0
400 MHz spectrometer using deuterated solvents. Tetramethylsilane (TMS) was used as
1
an internal reference (0 ppm) for H spectra recorded in CDCl
3
and the residual signal
, the
13
of the solvent (77.16 ppm) for C spectra. For NMR spectra recorded in DMSOꢀ
d
6
1
13
residual signal of the solvent was used to reference H and C chemical shifts.
Chemical shifts are reported in part per million (ppm) in the scale, coupling constants
in Hz and multiplicity as follows: s (singlet), d (doublet), t (triplet), q (quadruplet), qt
δ
(
quintuplet), m (multiplet), dd (doublet of doublets), td (doublet of triplets), ddd
doublet of doublet of doublets), br (broad signal). 2DꢀNOESY spectra were acquired in
(
6
DMSOꢀd with mixing times of 300 and 500 ms, and 2DꢀEXSY experiments were
carried out with the conventional NOESY pulse sequence using a mixing time of 1 s.
The proton signals of the
E and Z rotamers were identified by simple inspection of the
1
H spectrum and the rotamer ratio was calculated by peak integration. Exchange rate
(
kex) was estimated by plotting the intensity ratio for exchange crossꢀpeaks and the
25
corresponding diagonal peaks in NOESY spectra as a function of the mixing time.
Electrospray ionization mass spectra (ESIꢀMS) were recorded on an instrument
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equipped with single quadrupole detector coupled to an HPLC, and highꢀresolution
(HR) ESIꢀMS on a LC/MSꢀTOF instrument. A TOF analyser was used for mass spectra
obtained via electron impact ionization (EI), and values in parentheses indicate relative
intensities with regard to the base peak.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthesis of arylacetonitrile derivatives (11-15).
2-(2-Cyano-4-nitrophenyl)acetonitrile (11). A modified method was followed to
26
synthesize compound 11. 2ꢀcyanophenylacetonitrile (5 g, 35.2 mmol) was dissolved in
concentrated sulfuric acid (2 mL) cooled at 0ºC. Then, a solution of nitric acid (1.5 mL)
in sulfuric acid (7.1 mL) was added dropwise within 40 min, so that the temperature of
the reaction mixture did not exceed 5ºC. After stirring for 2.5 h at ꢀ5ºC, the reaction
mixture was poured over ice and treated with sodium carbonate until pH 8ꢀ9 was
reached. The solid was filtered, washed with water and dried in vacuum. After
purification by column chromatography (silica gel, from 50% hexane in DCM to 100%
DCM, then to 0.1% of MeOH in DCM), a yellow solid was obtained (3.96 g, 60%): mp
1
1
08ꢀ110 ºC; TLC: Rf (DCM) 0.34; H NMR (400 MHz, CDCl ) δ (ppm): 8.58 (1H, d,
3
J
=2.4 Hz), 8.54 (1H, dd,
J
=8.8 Hz,
J=2.4 Hz), 7.95 (1H, d, J=8.8 Hz), 4.13 (2H, s);
1
3
1
C{ H} NMR (101 MHz, CDCl
3
) δ (ppm): 147.7, 140.0, 130.4, 128.3, 128.1, 114.6,
+
1
14.5, 113.8, 22.9; MS (EI): m/z 187 (M , 97), 141 (100), 114 (84) (calcd mass for
+
C
9
H
5
N
3
O
2
[M] : 187).
2-(2,4-Dinitrophenyl)acetonitrile (12). The published method with some modifications
27
was followed to synthesize compound 12. N,Nꢀdimethylformamide dimethyl acetal
71 mL, 530 mmol ) was added to a solution of 2,4ꢀdinitrotoluene (20 g, 106 mmol) in
(
dry DMF (150 mL). The reaction mixture was stirred at 160ºC under an Ar atmosphere
for 15 h. Then, the solvent was removed under vacuum. The crude was dissolved in
DCM and precipitated upon addition of cold hexane to give (E)ꢀ2ꢀ(2,4ꢀdinitrophenyl)ꢀ
N,Nꢀdimethylꢀ1ꢀethenamine (16) as a red solid (24.3 g, 96%), which was used without
1
further purification. Characterization for 16: H NMR (400 MHz, CDCl
3
,) δ (ppm): 8.73
=9.2 Hz), 7.31 (1H,
=13 Hz), 3.06 (6H, s). Compound 16 (24.2 g, 102 mmol) and
(
1H, d,
J=2.4 Hz), 8.04 (1H, dd, J=9.2 Hz, J=2.4 Hz), 7.49 (1H, d, J
d, =13 Hz), 6.04 (1H, d,
J
J
hydroxylamine hydrochloride (10.6 g, 153 mmol) were dissolved in formic acid (150
mL) and the reaction mixture was stirred at 110ºC for 5 h. After removal of the solvent
under vacuum, the crude was dissolved in toluene and evaporated again under reduced
pressure. The crude was dissolved in DCM (100 mL) and washed with saturated
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NaHCO
3
(3x50 mL) and brine (1x50 mL). The combined organic layers were dried over
4
anhydrous MgSO and filtered. The solvent was removed under vacuum and the product
was purified by column chromatography (silica gel, 50ꢀ100% of DCM in hexane). The
appropriate fractions were collected and the solvents were removed under vacuum to
give of a brown solid, which was used without further purification (12.6 g, 60%): mp
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
91ꢀ92 ºC; TLC: Rf (DCM) 0.70; H NMR (400 MHz, CDCl ) δ (ppm): 9.03 (1H, d,
3
J
=2.4 Hz), 8.57 (1H, dd, J=8.4 Hz, J=2.4 Hz), 8.04 (1H, d, J=8.4 Hz), 4.34 (2H, s);
1
3
1
C{ H}NMR (101 MHz, CDCl
3
) δ (ppm): 148.2, 147.8, 132.7, 132.2, 128.5, 121.4,
+
115.2, 23.1; MS (EI): m/z 207 (M , 2), 180 (100), 134 (95), 114 (58) (calcd mass for
+
C
8
H
5
N
3
O
4
[M] : 207).
Ethyl 2-cyano-2-(2-fluoro-4-nitrophenyl)acetate (17). The published method with some
28
modifications was followed to synthesize compound 17. 1,2ꢀdifluoroꢀ4ꢀnitrobenzene
(5 g, 31.4 mmol ) was added to a solution of KI (56 mg, 0.33 mmol), ethyl cyanoacetate
(3.9 g, 34.5 mmol) and K
2 3
CO (6.0 g, 44.0 mmol), finely blended, in anhydrous DMF
(50 mL). The reaction mixture was stirred first at room temperature for 16 h, and then at
100ºC for 2 h. After removal of the solvent under vacuum, the crude was partitioned
between aqueous citric acid 1 M (75 mL) and ethyl acetate (75 mL), and the layers were
separated. The aqueous layer was washed with ethyl acetate (50 mL) two times. Finally,
the combined organic layers were washed with brine (50 mL), dried over anhydrous
MgSO
4
and filtered. After removal of the solvent, a purple solid were obtained and used
1
without further purification (7.75 g, 98%); TLC:
CDCl ) δ (ppm): 8.15 (1H, ddd, J=8.8 Hz, = 2.4 Hz,
Hz, =2.4 Hz), 7.77 (1H, dd, =7.2 Hz), 5.09 (1H, s), 4.32 (2H, qd,
Hz), 1.33 (3H, t,
R
f
(DCM) 0.80; H NMR (400 MHz,
3
J
J
=1.2 Hz), 8.04 (1H, dd,
J
J
=9.2
=7.2
J
J
=8.4 Hz, J
1
3
1
J
=7.2 Hz); C{ H}NMR (101 MHz, CDCl
3
) δ (ppm): 162.7, 159.6 (d,
=2.5 Hz), 124.8 (d, =15 Hz), 120.0 (d,
=26.6 Hz), 64.3, 37.3 (d, =3.2 Hz), 13.9; F NMR (375
) δ (ppm): ꢀ111.2 (1F, m).
2-(2-Fluoro-4-nitrophenyl)acetonitrile (14). The published method with some
J
J
=256 Hz), 149.5 (d,
J
=8.7 Hz), 130.8 (d,
J
J
19
=3.8 Hz), 113.7, 112.0 (d,
J
J
MHz, CDCl
3
28
modifications was followed to synthesize compound 14. Compound 17 (7.7 g, 30.5
mmol ) was dissolved in a mixture of water/acetic acid (22.5 mL/ 7.5 mL) and then
37% HCl (4 mL) was added. The solution was stirred and heated at 100ºC for 12 h.
After quenching with saturated NaHCO (100 mL), the reaction was extracted three
3
times with diethyl ether (40 mL). The combined organics layers were collected and
4
washed with brine (50 mL), dried over anhydrous MgSO and filtered. Finally, the
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solvent was removed under vacuum. The product was purified by column
chromatography (silica gel, DCM). The appropriate fractions were collected and the
solvent was removed to give a purple solid (1.73 g, 32%): mp 79ꢀ81 ºC; TLC:
R
f
(DCM)
=2.2
) δ (ppm): 157.1 (d,
=15.7 Hz), 119.9 (d, =3.8 Hz),
): δ (ppm): ꢀ
1
0.45; H NMR (400 MHz, CDCl
3
) δ (ppm): 8.12 (1H, m), 8.01 (1H, dd,
J
=9.4 Hz,
J
1
3
1
Hz), 7.71 (1H, m), 3.89 (2H, s); C{ H}NMR (101 MHz, CDCl
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
J
=255 Hz), 148.9 (d,
J
=7.1 Hz), 130.4, 124.8 (d,
J
J
1
9
115.3, 111.6 (d,
J
=26 Hz), 17.9 (d,
J
=5 Hz); F NMR (375 MHz, CDCl
3
+
1
12.0 (1F, m); MS (EI): m/z 180 (M , 100), 134 (66), 107 (84) (calcd mass for
+
C H FN O [M] : 180).
8
5
2
2
Ethyl-2-cyano-2-(2,6-difluoro-4-nitrophenyl)acetate (18). A modified method was
28
followed to synthesize compound 18. 1,2,3ꢀtrifluoroꢀ5ꢀnitrobenzene (4.8 g, 27.1
mmol) was added to a solution of KI (44.7 mg, 0.26 mmol), ethyl cyanoacetate (3.4 g,
52.2 mmol), and K
mL). The reaction mixture was stirred first at room temperature for 20 h, and then at
00ºC for 2 h. After removal of the solvent under vacuum, the crude was partitioned
2 3
CO (5.28 g, 38.2 mmol), finely blended, in anhydrous DMF (90
1
between citric acid 1 M (75 mL) and ethyl acetate (75 mL). The aqueous layer was
washed with ethyl acetate (2x50 mL). Finally, the combined organic layers were washed
4
with brine (50 mL), dried over anhydrous MgSO and filtered. After removal of the
solvent, a yellow solid was obtained (5.3 g, 72%): TLC:
R
f
(20% hexane in DCM) 0.75;
=6.8 Hz), 5.16 (1H, s), 4.36 (2H,
=7.2 Hz); C{ H}NMR (101 MHz, CDCl ) δ (ppm): 162.1,
=6.7 Hz), 159.1 (d, =6.7 Hz), 114.6 (t, =18.3 Hz), 112.4, 108.4 (d, =24.3
Hz), 64.6, 31.9, 13.9; F NMR (375 MHz, CDCl ) δ (ppm): ꢀ106.8 (2F, d, =6.8 Hz).
-(2,6-Difluoro-4-nitrophenyl)acetonitrile (15). A modified method was followed to
1
H NMR (400 MHz, CDCl
=7.2 Hz), 1.35 (3H, t,
161.6 (d,
3
) δ (ppm): 7.93 (2H, d, J
13
1
qd,
J
J
3
J
J
J
J
19
3
J
2
2
8
synthesize compound 15. Compound 18 (5.3 g, 19.5 mmol ) was dissolved in a
mixture of water/acetic (22.5 mL/ 7.5 mL) and then 37% HCl (4 mL) was added. The
3
solution was stirred at 100ºC for 12 h. After quenching with saturated NaHCO (100
mL), the solution was extracted three times with diethyl ether (40 mL). The combined
organics layers were collected and washed with brine (50 mL), dried over anhydrous
4
MgSO and filtered. Finally, the solvent was removed under vacuum. The product was
purified by column chromatography (silica gel, DCM). The appropriate fractions were
collected and the solvent was removed to give a yellow solid (1.5 g, 39%): mp 85ꢀ86
1
ºC; TLC:
(2H, d,
R
f
(20% hexane in DCM) 0.75; H NMR (400 MHz, CDCl
3
) δ (ppm): 7.91
13
1
J
=6.8 Hz), 3.83 (2H, s); C{ H}NMR (101 MHz, CDCl
3
) δ (ppm): 159.8 (d,
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5
5
5
5
5
5
5
6
J
=254 Hz), 149.0 (d,
=3.9 Hz), 115.5, 111.7 (d,
) δ (ppm): ꢀ107.8 (1F, d,
(53) (calcd mass for C
J
=8.6 Hz), 130.6 (d,
J=3.3 Hz), 125.0 (d, J=15.8 Hz), 120.1 (d,
19
J
J
=26.3 Hz), 18.1 (d,
J
=4.9 Hz). F NMR (375 MHz,
+
CDCl
3
J
=6.8 Hz); MS (EI): m/z 198 (M , 100), 152 (66), 125
+
8
H
4
F
2
N
2
O
2
[M+H] : 198).
0
1
2
3
4
5
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7
8
9
0
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2
3
4
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8
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8
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9
0
Synthesis of model coumarin derivatives (3-9)
7
-(N,N-Diethylamino)-4-methyl-2-thiocoumarin (2). A modified method was followed
29
to synthesize compound 2. Coumarin 1 (1.72 g, 7.42 mmol) and Lawesson’s reagent
(1.65 g, 4.08 mmol) were dissolved in toluene (40 mL) and heated at 100ºC for 12 h.
After evaporation under reduced pressure, the dark residue was purified by column
chromatography (silica gel, DCM) to give an orange solid (1.70 g, 92%): TLC:
R
f
1
(DCM) 0.50; H NMR (400 MHz, CDCl
3
) δ (ppm): 7.44 (1H, d,
=7.2 Hz), 2.29 (3H, d,
=7.2 Hz); C{ H}NMR (101 Hz, CDCl ) δ (ppm): 197.0, 158.9, 150.9, 146.2,
125.6, 123.6, 111.2, 110.1, 97.2, 44.9, 17.8, 12.4; HRMS (ESIꢀTOF) m/z: [M+H]
Calcd for C H NOS 248.1109; Found 248.1099.
J
=9.6 Hz), 6.93 (1H, br
q,
J
=0.8 Hz), 6.69ꢀ6.65 (2H, m), 3.43 (4H, q,
J
J
=0.8 Hz), 1.24
13
1
(6H, t,
J
3
+
14 18
2
-(Dicyanomethylene)-7-(N,N-diethylamino)-4-methylcoumarin (
3
). A modified method
2
9
was followed to synthesize compound 3. Silver nitrate (172 mg, 1.02 mmol) was
added to a solution of 2 (100 mg, 0.40 mmol), malononitrile (40 mg, 0.61 mmol) and
triethylamine (200 ꢀL, 1.41 mmol) in dry ACN (20 mL) under an argon atmosphere.
The reaction mixture was stirred for 3 h in the dark at room temperature and then
concentrated under reduced pressure, The crude product was purified by column
chromatography (silica gel, 50ꢀ100% of DCM in hexane) to give a yellow solid (97.6
1
mg, 86%); mp 187ꢀ189 ºC; TLC:
7.42 (1H, d, =9.2 Hz), 6.68 (1H, dd,
q, =7.2 Hz), 2.41 (3H, d, =0.7 Hz), 1.24 (6H, t,
CDCl ) δ (ppm): 171.9, 154.9, 151.6, 149.9, 126.0, 115.2, 114.4, 110.4, 110.0, 108.6,
97.1, 44.9, 18.6, 12.5; HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C H N O 280.1450;
R
f
(DCM) 0.53; H NMR (400 MHz, CDCl
3
) δ (ppm):
J
J=9.2 Hz, J=2.6 Hz), 6.59ꢀ6.57 (2H, m), 3.45 (4H,
13
1
J
J
J=7.2 Hz); C{ H}NMR (101 Hz,
3
+
17
18
3
Found 280.1440.
General procedure for the synthesis of coumarin derivatives 4-9: Compound 2 (150 mg,
0.61 mmol) was added to a solution of the corresponding arylacetonitrile derivative (10-
5) (0.92 mmol, 1.5 mol equiv.) in anhydrous ACN (10 mL) under an Ar atmosphere
and protected from light. After addition of triethylamine (126 L, 0.92 mmol, 1.5 mol
1
ꢀ
equiv.) the reaction mixture, which acquired an intense dark colour, was stirred for 2 h.
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Then, silver nitrate (226 mg, 1.34 mmol, 2.2 mol equiv.) was added and the reaction
mixture was stirred at room temperature for 1ꢀ2 h under an Ar atmosphere and protected
from light. The crude product was evaporated under reduced pressure and purified by
column chromatography (silica gel, in most cases the following gradient was used: 0ꢀ
100% DCM in hexane) to give the desired coumarin derivative (4-9).
10
11
12
13
14
15
16
17
18
19
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2
-(Cyano(4-nitrophenyl)methylene)-7-(N,N-diethylamino)-4-methylcoumarin (4). Dark
1
red solid (91 mg, 40%): mp 228ꢀ230 ºC; TLC:
R
f
(DCM) 0.8; H NMR (400 MHz,
CDCl
3
) δ (ppm): (major rotamer) 8.25 (2H, d,
J
=9.2 Hz), 7.99 (2H, d,
=8.8 Hz, =2.4 Hz), 6.43 (1H, d,
=1.2 Hz), 1.24 (6H, t, =7.2 Hz);
J=9.2 Hz), 7.34
(1H, d,
J
=8.8 Hz), 6.77 (1H, br q), 6.60 (1H, dd,
J
J
J
=2.4 Hz), 3.45 (4H, q,
J
=7.2 Hz), 2.36 (3H, br d,
J
J
1
3
1
C{ H}NMR (101 Hz, CDCl
3
) δ (ppm): (major rotamer) 162.9, 154.3, 150.7, 144.7,
1
44.3, 140.4, 126.8, 125.5, 123.8, 120.1, 112.3, 110.1, 109.3, 96.8, 82.1, 44.7, 18.4,
+
12.5; HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C22
H
22
N
3
O
3
376.1656; Found
3
76.1658; Analytical HPLC (50 to 100% B in 30 min: R
t
= 26.1 min).
2-(Cyano(2-cyano-4-nitrophenyl)methylene)-7-(N,N-diethylamino)-4-methylcoumarin
1
(5
). Dark red solid (132 mg, 54%): mp 230ꢀ234 ºC (decomp.); TLC:
NMR (400 MHz, CDCl ) δ (ppm): (major rotamer) 8.58 (1H, d, =2.4 Hz), 8.41 (1H,
dd, =8.8 Hz, =2.4 Hz), 7.79 (1H, d, =8.8 Hz), 7.36 (1H, d, =9.2 Hz), 6.73 (1H, br q,
=1.2 Hz), 6.59 (1H, dd, =9.2 Hz, =2.6 Hz), 6.31 (1H, d,
=7.2 Hz), 2.39 (3H, d, =0.8 Hz), 1.20 (6H, t,
f
R (DCM) 0.45; H
3
J
J
J
J
J
J
J
J
J
J
=2.6 Hz), 3.41 (4H, q,
1
3
1
J
J
=7.2 Hz); C{ H}NMR (101 MHz,
3
CDCl ) δ (ppm): (major rotamer) 164.1, 154.0, 151.0, 146.9, 145.2, 143.6, 132.4, 131.0,
129.2, 127.2, 125.8, 119.4, 116.6, 112.6, 110.9, 110.1, 109.6, 97.1, 44.8, 18.5, 12.4;
+
HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C23
H
21
N
4
O
3
401.1608; Found 401.1610;
t
Analytical HPLC (50 to 100% B in 30 min: R = 19.9 min).
2-(Cyano(2,4-dinitrophenyl)methylene)-7-(N,N-diethylamino)-4-methylcoumarin
(6).
1
Dark violet solid (175 mg, 68%): mp 208ꢀ209 ºC; TLC: Rf (DCM) 0.56; H NMR (400
MHz, CDCl
(1H, d, =8.8 Hz), 7.33 (1H, d,
=2.4 Hz), 6.23 (1H, d, =2.4 Hz), 3.42 (4H, q,
=7.2 Hz) ; C{ H}NMR (101 Hz, CDCl ) δ (ppm): 163.7, 153.9, 151.0, 147.3, 146.9,
3
) δ (ppm): 8.75 (1H, d,
=8.8 Hz), 6.70 (1H, br q), 6.58 (1H, dd,
=7.2 Hz), 2.37 (3H, br s), 1.24 (6H, t,
J
=2.4 Hz), 8.38 (1H, dd,
J
=8.8 Hz,
J
= 2.4 Hz), 7.86
J
J
J
=8.8 Hz,
J
J
J
J
1
3
1
3
144.9, 134.2, 131.7, 126.6, 125.7, 121.0, 119.4, 111.0, 110.0, 109.6, 96.9, 44.9, 18.5,
+
1
2.4; HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C H N O 421.1506; Found
22
21
4
5
4
t
21.1506; Analytical HPLC (50 to 100% B in 30 min: R = 21.8 min).
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2
2
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3
3
3
3
3
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3
4
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5
5
5
5
5
5
5
6
2-(Cyano(2,4,6-trinitrophenyl)methylene)-7-(N,N-diethylamino)-4-methylcoumarin (7).
Dark green solid (50 mg from 100 mg of 2, 27%); mp 223ꢀ224 ºC; TLC:
R
f
(2% MeOH
=9.2
=2.4 Hz), 3.41
J=7.2 Hz); C{ H}NMR (101 Hz,
1
in DCM) 0.8; H NMR (400 MHz, CDCl
3
) δ (ppm): 8.85 (2H, s), 7.37 (1H, d,
=9.2 Hz, =2.4 Hz), 6.16 (1H, d,
=7.2 Hz), 2.39 (3H, br s), 1.23 (6H, t,
J
Hz), 6.71 (1H, br s), 6.61 (1H, dd,
4H, q,
J
J
J
1
3
1
(
J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CDCl ) δ (ppm): 164.6, 153.8, 151.2, 149.7, 148.5, 144.8, 128.0, 126.0, 123.2, 116.4,
3
+
110.2, 110.1, 110.0, 96.7, 71.1, 45.0, 18.6, 12.4; HRMS (ESIꢀTOF) m/z: [M+H] Calcd
20 5 7
for C22H N O 466.1357; Found 466.1362; Analytical HPLC (50 to 100% B in 30 min:
R = 19.7 min).
t
2-(Cyano(2-fluoro-4-nitrophenyl)methylene)-7-(N,N-diethylamino)-4-methylcoumarin
1
(8
). Dark red solid (101 mg, 42%): mp 159ꢀ160 ºC; TLC:
R
f
(DCM) 0.53; H NMR (400
MHz, CDCl ) δ (ppm): (major rotamer) 8.07 (1H, ddd,
J
=8.6 Hz,
=8.6 Hz, =7.2 Hz), 7.32 (1H, d,
=8.9 Hz, =2.5 Hz), 6.24 (1H, d,
=1.2 Hz), 1.19 (6H, t, =7.2 Hz);
) δ (ppm): (major rotamer) 163.4, 158.9 (d, =256 Hz),
=8.5 Hz), 144.50, 130.9 (d, =3.4 Hz), 128.5 (d, =14.3 Hz),
=3.4Hz), 112.0, 111.8, 111.3, 110.0, 109.0, 96.9, 74.6, 44.6,
J=2.4 Hz, J=0.8 Hz),
3
8.01 (1H, dd,
J
=10.0 Hz,
J
= 2.4 Hz), 7.73 (1H, dd,
=1.2 Hz), 6.56 (1H, dd,
=7.2 Hz), 2.34 (3H, d,
J
J
J
J
=8.9 Hz), 6.72 (1H, br q,
J
J
J
=2.5 Hz), 3.39 (4H, q,
J
J
J
1
3
1
C{ H}NMR (101 MHz, CDCl
3
J
1
54.4, 150.6, 146.7 (d,
25.4, 119.5, 119.2 (d,
J
J
J
1
J
1
9
18.3, 12.5; F NMR (375 MHz, CDCl
=10.0 Hz,
3
) δ (ppm): (major rotamer) ꢀ104.3 (1F, dd,
+
J
J
= 7.2 Hz); HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C22
H
21FN
3
O
3
3
94.1561; Found 394.1566; Analytical HPLC (50 to 100% B in 30 min: R
t
= 23.8 min).
2-(Cyano(2,6-difluoro-4-nitrophenyl)methylene)-7-(N,N-diethylamino)-4-
methylcoumarin (
9
). Dark red solid (137 mg, 55%): mp 168ꢀ169 ºC; TLC:
) δ (ppm): (major rotamer) 7.87 (2H, d,
=8.8 Hz), 6.73 (1H, br q, =1.2 Hz,), 6.56 (1H, dd, =8.8 Hz,
=7.2 Hz), 2.35 (3H, d, =1.2 Hz), 1.18 (6H, t,
) δ (ppm): (major rotamer) 164.4, 160.0
=11 Hz), 144.9, 125.5, 118.3, 117.7
R
f
(DCM)
=7.2 Hz),
=2.4 Hz),
1
0
.64; H NMR (400 MHz, CDCl
3
J
7.33 (1H, d,
J
J
J
J
6.18 (1H, d,
J
=2.4 Hz), 3.38 (4H, q,
J
J
13
1
J
=7.2 Hz); C{ H}NMR (101 MHz, CDCl
3
(dd,
J
=256 Hz,
J
=7.3 Hz), 154.6, 150.6, 147.1 (t,
J
1
9
(t,
J
=19 Hz), 110.7, 109.8, 109.0, 107.9, 107.6, 96.9, 64.7, 44.6, 18.3, 12.4; F NMR
+
3
(375 MHz, CDCl ) δ (ppm): ꢀ102.7 (2F, d, J=7.2 Hz); HRMS (ESIꢀTOF) m/z: [M+H]
Calcd for C22
20 2 3 3
H F N O 412.1467; Found 412.1470; Analytical HPLC (50 to 100% B
in 30 min: R = 23.7 min).
t
Synthesis of coumarin-caged derivatives of benzoic acid (25-28).
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4-(Benzoyloxymethyl)-7-(N,N-diethylamino)-2-thiocoumarin (24). A solution of HCl in
21
1
,4ꢀdioxane (4 M, 2.5 mL, 10 mmol) was added to 22 (1.5 g, 4.92 mmol) in absolute
ethanol (120 mL). The reaction mixture was heated at 90ºC for 20 h and evaporated
under vacuum. The resulting orange solid was dissolved in dry DCM (25 mL) at 0ºC
under an Ar atmosphere together with 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hydrochloride (0.94 g, 4.9 mmol), DMAP (0.60 g, 4.9 mmol) and benzoic acid (0.72 g,
5.9 mmol). After 10 min at 0ºC, the reaction mixture was stirred at room temperature
for 18 h in the dark and under Ar. Subsequently, the organic solution was washed with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aqueous saturated NaHCO (1 x 20 mL), aqueous 10% ammonium chloride (2 x 25
3
2 4
mL), water (1 x 25 mL) and dried over anhydrous Na SO . After purification by column
chromatography (silica gel, DCM), an orange solid was obtained (1.3 g, 72%): TLC:
R
f
1
(
DCM) 0.8; H NMR (400 MHz, CDCl ) δ (ppm): 8.11 (2H, m, HAr), 7.64ꢀ7.60 (1H,
3
m), 7.51ꢀ7.43 (3H, m, HAr), 7.15 (1H, t, H3, J= 1.2 Hz), 6.71ꢀ6.67 (2H, m, HAr), 5.43
(2H, d, CH
2 2 3
, J= 1.2 Hz), 3.44 (4H, q, CH Et, J= 7.2 Hz), 1.23 (6H, t, CH Et, J 7.2 Hz);
1
3
1
C{ H}NMR (101 MHz, CDCl ) δ (ppm): 197.3, 166.0, 159.2, 151.2, 142.1, 138.8,
3
130.0, 129.2, 128.7, 124.7, 120.9, 110.5, 108.4, 97.6, 61.7, 45.1, 12.6; HRMS (ESIꢀ
+
TOF) m/z: [M+H] Calcd for C21
H22NO
3
S 368.1320; Found 368.1312.
General procedure for the synthesis of coumarin derivatives 25-27
Compound 24 (150 mg, 0.41 mmol) and the corresponding arylacetonitrile derivative
(10, 12 or 13) (0.61 mmol, 1.5 mol equiv. for 10 and 12; 0.82 mmol, 2 mol equiv. for
1
3) were dissolved in anhydrous ACN (20 mL) (25, 26) or in a 1:1 mixture of
anhydrous DCM/ACN (20 mL) (27) under an Ar atmosphere and protected from light.
After addition of triethylamine (25, 26: 86 L, 0.61 mmol, 1.5 mol equiv.; 27: 113 L,
ꢀ
ꢀ
0.82 mmol, 2 mol equiv.), the reaction mixture, which acquired an intense dark colour,
was stirred for 2 h. Then, silver nitrate (153 mg, 0.9 mmol, 2.2 mol equiv.) was added
and the reaction mixture was stirred at room temperature for 2 h under an Ar
atmosphere and protected from light. The crude product was evaporated under reduced
pressure and purified by column chromatography (silica gel, 0ꢀ100% DCM in hexane)
to give the desired coumarin derivative (25-27).
4-(Benzoyloxymethyl)-2-(cyano(4-nitrophenyl)methylene)-7-(N,N-
diethylamino)coumarin (25). Red solid (140 mg, 69%): mp 219ꢀ222 ºC (decomp.);
1
TLC: R
f
(DCM) 0.44; H NMR (400 MHz, CDCl
3
) δ (ppm): (major rotamer) 8.27 (2H,
m), 8.12 (2H, m), 7.99 (2H, m), 7.63ꢀ7.57 (1H, m), 7.51ꢀ7.46 (2H, m), 7.36 (1H, d, J=
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8.8 Hz), 7.08 (1H, br t), 6.59 (1H, dd,
2H, d, =1.2 Hz), 3.45 (4H, q, =7.2 Hz), 1.24 (6H, t,
MHz, CDCl ) δ (ppm): (major rotamer) 166.0, 162.1, 154.5, 150.8, 145.2, 140.5, 139.8,
J=8.8 Hz, J=2.4 Hz), 6.44 (1H, d, J=2.4 Hz), 5.42
13
1
(
J
J
J=7.2 Hz); C{ H}NMR (101
3
133.6, 129.8, 129.2, 128.7, 127.2, 124.8, 123.9, 119.4, 111.4, 109.4, 107.3, 97.1, 84.7,
+
6
2.3, 44.7, 12.5; HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C29
H
26
3
N O
5
496.1867;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Found 496.1857; Analytical HPLC (50 to 100% B in 30 min: R = 27.9 min).
t
4-(Benzoyloxymethyl)-7-(N,N-diethylamino)-2-(cyano(2,4-dinitrophenyl)methylene)-
coumarin (26). Dark violet solid (90 mg, 41%): mp 220ꢀ223 ºC (decomp.); TLC:
R
f
1
(
DCM) 0.5; H NMR (400 MHz, CDCl ) δ (ppm): 8.77 (1H, d,
J
=2.4 Hz), 8.41 (1H, dd,
=8.8 Hz), 7.62 (1H, m), 7.50 (2H, m),
=8.8 Hz, =2.4 Hz), 6.26 (1H, d, =2.4
=7.2 Hz), 1.24 (6H, t, =7.2 Hz);
) δ (ppm): 165.9, 163.0, 154.1, 151.1, 147.6, 145.4,
3
J
=8.8 Hz,
7.34 (1H, d, H5), 7.01 (1H, br t), 6.57 (1H, dd,
Hz), 5.43 (2H, d, =0.8 Hz), 3.42 (4H, q,
C{ H}NMR (101 MHz, CDCl
J= 2.4 Hz), 8.12 (2H, m), 7.89 (1H, d, J
J
J
J
J
J
J
13
1
3
142.8, 133.6, 131.9, 129.8, 129.1, 128.7, 126.7, 124.9, 120.9, 118.7, 109.8, 109.4,
+
1
07.1, 97.2, 79.4, 62.1, 45.0, 12.4; HRMS (ESIꢀTOF) m/z: [M+H] Calcd for
29 25 4 7 t
C H N O 541.1718; Found 541.1718; Analytical HPLC (50 to 100% B in 30 min: R
= 23.2 min).
-(Benzoyloxymethyl)-2-(cyano(2,4,6-trinitrophenyl)methylene)-7-(N,N-
4
diethylamino)coumarin (27). Dark green solid (19 mg, 8%): mp 222ꢀ224 ºC (decomp.);
1
TLC:
R
f
(DCM) 0.4; H NMR (400 MHz, CDCl
3
) δ (ppm): 8.89 (2H, s), 8.13 (2H, m),
7
.62 (1H, m), 7.50 (2H, m), 7.36 (1H, d,
J
=9.2 Hz), 7.03 (1H, br t), 6.61 (1H, dd,
J
J
=9.2
=7.2
Hz,
J
=2.4 Hz), 6.18 (1H, d,
J
=2.4 Hz), 5.45 (2H, br d, =0.8 Hz), 3.41 (4H, q,
J
1
3
1
Hz), 1.23 (6H, t,
J
=7.2 Hz); C{ H}NMR (101 MHz, CDCl
3
) δ (ppm): 165.8, 164.1,
154.0, 151.2, 149.9, 145.4, 144.2, 133.7, 129.9, 129.0, 128.7, 127.4, 125.1, 123.2,
+
115.9, 110.2, 108.3, 107.2, 97.0, 73.4, 61.9, 45.0, 12.4; HRMS (ESIꢀTOF) m/z: [M+H]
Calcd for C H N O 586.1569; Found 586.1560; Analytical HPLC (50 to 100% B in
29
24
5
9
30 min: R
t
= 20.9 min).
4-(Benzoyloxyeth-1-yl)-7-(N,N-diethylamino)-2-(cyano(4-
nitrophenyl)methylene)coumarin (28). A solution of 4ꢀ(benzoyloxymethyl)ꢀ7ꢀ(N,N
ꢀ
21
diethylamino)ꢀ4ꢀ(1ꢀhydroxyethꢀ1ꢀyl)ꢀ2ꢀthiocoumarin (75 mg, 0.20 mmol) was added
to a solution of 2ꢀnitrophenylacetonitrile (95.5 mg, 0.59 mmol) and NEt (82 µL, 0.59
3
mmol) in a 1:1 (v/v) mixture of anhydrous ACN and DCM (4 mL) under an Ar
atmosphere. After stirring for 2.5 h at room temperature in the dark, silver nitrate (73.5
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mg, 0.43 mmol) was added and the reaction mixture was stirred for additional 1.5 h.
After evaporation under reduced pressure, the crude product was purified by column
chromatography (silica gel, hexane/AcOEt 6:4) to give a dark red solid (69 mg, 69%);
1
mp 219ꢀ221 ºC (decomp.); TLC:
CDCl ) δ (ppm): (major rotamer) 8.26 (2H, d,
2H, d, =9.0 Hz), 7.61 (1H, t, =7.2 Hz), 7.51ꢀ7.45 (3H, m), 7.08 (1H, br s), 6.60 (1H,
=9.2 Hz, =2.4 Hz), 6.44 (1H, d,
R
f
(2% MeOH in DCM) 0.90; H NMR (400 MHz,
3
J=9.0 Hz), 8.13 (2H, m, J=8.0 Hz), 7.98
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
J
J
dd,
J
J
J
=2.4 Hz), 6.30 (1H, q,
J
= 6.8 Hz), 3.45 (4H, q,
13
1
J
=7.2 Hz), 1.75 (3H, s,
J
=6.8Hz), 1.24 (6H, t,
J
=7.2 Hz); C{ H}NMR (101 MHz,
CDCl ) δ (ppm): (major rotamer) 163.7, 160.5, 152.9, 148.8, 144.8, 143.2, 138.1, 131.6,
3
127.9, 127.6, 126.8, 126.7, 125.2, 123.0, 122.0, 117.7, 107.5, 107.2, 104.9, 95.3, 82.4,
+
6
6.3, 42.8, 19.0, 10.6; HRMS (ESIꢀTOF) m/z: [M+H] Calcd for C30
28
H N
3
O
5
510.2023;
Found 510.2019; Analytical HPLC (50 to 100% B in 30 min: R = 29.2 min).
t
Photophysical properties.
Absorption spectra were recorded in a Varian Cary 500 UVꢀVisꢀNIR spectrophotometer
at 25 ºC. Emission spectra were registered in a Photon Technology International (PTI)
fluorimeter. Solutions for fluorescence quantum yield measurements were adjusted to
concentrations such that the absorption was around 0.04 at the corresponding excitation
wavelength. Fluorescence quantum yields, Φ
F
, were calculated from equation (1):
s
2
Abs A n
s
F
Φ = Φ
F
s
2
Abs A ns
(1)
in which the superscript “
excitation wavelength (λEx),
spectrum, and is the refractive index of the solvent used. The uncertainty in the
experimental value of Φ has been estimated to be ca. 10 %. The standard fluorophore
used for the determination of the fluorescence quantum yield was either 7ꢀdiethylaminoꢀ
ꢀmethylcoumarin (coumarinꢀ1, λEx = 360 nm) or fluorescein (λEx = 460 nm), with a
s” stands for standard samples, Abs is the absorbance at the
A
is the integrated area of the corresponding emission
22
n
F
4
F
reported absolute fluorescence quantum yield, Φ , of 0.73 (in ethanol) and 0.92 (in 0.1
23
M aqueous sodium hydroxide), respectively. All samples were measured in 1 × 1 cm
quartz cuvettes (Hellma).
Irradiation experiments.
Photolysis studies were performed at 37ºC in a customꢀbuilt irradiation setup from
Microbeam including a cuvette, thermostated cuvette holder and a mounted high power
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
LED of 505 nm (100 mW/cm ). In a typical experiment, the irradiation samples
contained the coumarinꢀcaged model compound (20
the samples were analyzed by reversedꢀphase HPLCꢀESI MS in a Jupiter Proteo C18
column (250x4.6 mm, 90 Å 4 m, flow rate: 1 mL/min) using linear gradients of 0.1%
ꢀM) in MeOH. After irradiation,
ꢀ
formic acid in H O (A) and 0.1% formic acid in ACN (B). The uncaging quantum yield
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
21
for coumarin derivatives 25 and 28 was estimated as previously described. Briefly, 1.7
mL of 20
ꢀM solutions of the compounds were irradiated in a 1 x 1 cm quarz cuvette at
ꢀ7
505 nm using a LED lamp. The irradiance was determined as 1.05 x 10 Einstein/s by
21
actinometry with Aberchrome 540.
ASSOCIATED CONTENT
Supporting Information
HPLC traces and UVꢀvis absorption and emission spectra of the compounds and results
1
3
19
from the photolysis experiments. 1D NMR (1H, C and F), MS and selected 2D NMR
spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: vmarchan@ub.edu
ACKNOWLEDGEMENTS
This work was supported by funds from the Spanish Ministerio de Economía y
Competitividad (grants CTQ2014ꢀ52658ꢀR and CTQ2015ꢀ65770ꢀP) and the Generalitat
de Catalunya (2014SGR187 and XRB). The authors acknowledge Dr. Francisco
Cárdenas (Unitat d’RMN, CCiTUB) for helpful assistance and fruitful discussions
during NMR characterization of the compounds, and Dr. Irene Fernández and Laura
Ortiz (CCiTUB) for MS characterization. A.G. was a recipient fellow of the University
of Barcelona.
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