New coumarin-urea based receptor for anions: A selective off-on fluorescence response to fluoride

Article abstract of DOI:10.1016/j.tet.2012.02.067

A novel fluorosensor for anions, 1,11-bis(4-methylcoumarin-7-yl)-6-methyl- 1,3,6,9,11-pentaazaundeca-2,10-dione (L), which contains two coumarin-urea units spaced by a flexible diethylenetriamine fragment, was easily prepared in 65% yield by a one-pot two

Full text of DOI:10.1016/j.tet.2012.02.067

Tetrahedron 68 (2012) 3768e3775
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
New coumarin-urea based receptor for anions: a selective offeon fluorescence
response to fluoride
,
a
a
b
Gianluca Ambrosi a, Mauro Formica a, Vieri Fusi a , Luca Giorgi , Eleonora Macedi , Giovanni Piersanti ,
*
Michele Retini b, Maria Antonietta Varrese a, Giovanni Zappia b
,
*
a Department of Base Sciences and Fundamentals (Chemistry Section), University of Urbino “Carlo Bo”, P.zza Rinascimento 6, I-61029 Urbino, Italy
b Department of Biomolecular Sciences, University of Urbino “Carlo Bo”, P.zza Rinascimento 6, I-61029 Urbino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel fluorosensor for anions, 1,11-bis(4-methylcoumarin-7-yl)-6-methyl-1,3,6,9,11-pentaazaundeca-
2,10-dione (L), which contains two coumarineurea units spaced by a flexible diethylenetriamine frag-
ment, was easily prepared in 65% yield by a one-pot two-step procedure. The binding ability of L toward
several anions (G) was evaluated by 1H NMR, UVevis, and fluorescence titration. It was found that L
forms stable [LG] and [LG2] species in both DMSO and CH3CN solvents. Furthermore, the fluorescence
emission of L in the visible range (400 nm) is affected by the presence of anions; it is quenched by ac-
etate, chloride, and pyrophosphate while it is enhanced by fluoride. Thus, this novel fluorosensor pro-
vides a selective offeon response to the presence of fluoride in solution.
Received 30 October 2011
Received in revised form 6 February 2012
Accepted 27 February 2012
Available online 8 March 2012
Keywords:
Coumarin
Fluoride
Fluorescence
Urea
Ó 2012 Elsevier Ltd. All rights reserved.
Anion coordination
1. Introduction
been designed to optimize and enrich this characteristic. Colori-
metric and/or fluorescent sensors for anions are particularly at-
Anions are ubiquitous and play important roles in many chem-
ical, environmental, and biological systems.1e7 Anions such as py-
rophosphate and ATP are involved in important biological and
metabolic processes and participate in various enzymatic re-
actions.8 Fluoride is commonly used in dental care applications and
exhibits inhibition of certain enzyme functions;9 however, dental
and skeletal fluorosis associated with high levels of fluoride in
drinking water have been reported in various countries.10 Recently,
fluoride has been associated with osteosarcoma,11 and exerting
some effects on the brain, such as lower IQ values in humans, in the
presence of high fluoride concentrations in water.12 High levels of
fluoride have also been correlated with the inhibition of neuro-
transmitter biosynthesis in fetuses.13 Researchers have focused
numerous efforts on the design and synthesis of host molecules able
to recognize and sense anions through optical and electrochemical
responses.14e18 An important class is represented by systems in
which the receptor is a neutral metal-free ligand and the directional
hydrogen bond is the predominant key to activate an interaction
with guests. Suitable molecular topologies, possessing functional
groups such as amides,19 ureas/thioureas,20 and pyrroles,21 have
tractive because they offer the potential for high sensitivity in the
presence of a low analyte concentration.22 These fluorescent sen-
sors are generally made up of two distinct fragments having two
distinct functions: one part is devoted to anion binding, while the
other is devoted to signaling the coordination by optical changes. In
addition, there is a growing interest23 in the immobilization of
anion receptors in polymeric materials, either as a pendant side-
chain or as an additive, with several advantages for small molecule
analogues and numerous potential applications in the environ-
mental and biological fields, especially in the case of fluorescent
anion sensors.
Recently, we have explored anion binding of a receptor family
based on macrocyclic amines with two urea moieties bearing the
signaling unit.24,25 These hosts are able to strongly interact with
acetate and halide anions in solution; and the anion signaling often
occurs in the UV range.25 Here, we describe the preparation and the
recognition studies of a novel fluorescent anion sensor, 1,11-bis(4-
methylcoumarin-7-yl)-6-methyl-1,3,6,9,11-pentaazaundeca-2,10-
dione, L in Fig. 1, as a model compound for the development of
materials in which L is attached as a side-chain appendage to
a polymeric framework.
The binding and recognition properties of L toward several
anions, including Fꢀ, Clꢀ, Brꢀ, AcOꢀ, HP2O37ꢀ, and OHꢀ were in-
vestigated in DMSO and CH3CN by measuring the optical response.
* Corresponding authors. Tel.: þ39 (0)722 303339; fax:þ39 (0)722 303313;
e-mail addresses: vieri.fusi@uniurb.it (V. Fusi), giovanni.zappia@uniurb.it (G. Zappia).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.02.067

G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
3769
displacement by a careful selection of the leaving groups. Indeed, 7-
amino-4-methylcoumarin 3 was reacted with the easy to handle
and store p-nitrophenylchloroformate31 5 in CH2Cl2 in the presence
of a slight excess (1.05 equiv) of pyridine at room temperature for
60 min to give carbamate 6 with an excellent isolated yield of 88%
(Scheme 1).
Notably, the above intermediate is very stable and can be pu-
rified by column chromatography or by recrystallization. The sub-
sequent addition of triamine 1 was carried out with activated 7-
amino-4-methylcoumarin 6 in CH2Cl2 in the presence of a slight
excess of pyridine. Under these reaction conditions, the desired
urea was obtained in very low yield; but simply replacing the
pyridine with N,N-diisopropylethylamine (DIPEA) and using DMF
as the solvent, the ligand L was obtained in 78% yield after flash
chromatography. Taking advantage of the above results, a one-pot
two-step procedure was then developed to obtain the desired tar-
get compound in a gratifying 65% overall yield as a white solid. The
overall reaction occurs under very mild conditions and avoids the
need to isolate and/or manipulate potentially toxic isocyanates.
Fig. 1. Structure of the new coumarineurea based receptor for anions.
2. Results and discussion
2.1. Synthesis
Unsymmetrically substituted urea derivatives have received
considerable attention due to their wide range of applications in
agriculture, petrochemicals, medicine, biology, as well as for their
use as intermediates in the preparation of organocatalysts, and their
synthesis has been recently reviewed.26e28 The conventional
methods reported for urea moiety synthesis are essentially based on
the use of phosgene or its synthetic equivalents.29 The aim of this
general approach is to generate in situ an isocyanate and, by sub-
sequent reaction30 with a different amine, to obtain N,N0-di-
substituted or N,N0,N00-trisubstituted unsymmetrical ureas.
Although this is an attractive and efficient strategy for the prepa-
ration of symmetrical ureas, the synthesis of unsymmetrically
substituted ureas has proven to be more challenging. Thus,
according to the above-mentioned synthetic approach, there are
two possibilities: either the isocyanate moieties shouldbe generated
from the triamine 1 or from 7-amino-4-methylcoumarin 3 (Scheme
1). Pivotal experiments showed that any attempt, using the most
common reagents,30 to prepare the aliphatic bis-isocyanate 2 from 1,
followed by the addition of phenylamine, gave negligible yields of
the corresponding bis-urea. On the other hand, any attempt to
generate 7-isocyanate-4-methyl coumarin 4 in situ using bis(4-
nitrophenyl)carbonate, triphosgene [bis(trichloromethyl)carbon-
ate], di-tert-butylcarbonate, or N,N0-carbonyldiimidazole (CDI)
failed, and the unreacted coumarin derivative 3 was recovered at the
end of the reaction. Only with triphosgene were traces of symmet-
rical urea formed after the addition of 1.
2.2. Solution studies
The binding properties of L with several anions G (G¼AcOꢀ, Fꢀ,
Clꢀ, Brꢀ, OHꢀ, HP2O37ꢀ) were studied by 1H NMR, UVevis, and
fluorescence techniques in both DMSO and CH3CN. The addition
constants of the adducts formed were determined by spectroscopic
titrations and are reported in Table 1. In CH3CN, the data were
obtained from UVevis and fluorescence titrations because L was
not soluble enough in this solvent for the NMR experiments; while,
it was possible to determine the addition constants also 1H NMR in
DMSO.
Table 1
Logarithmsd of the addition constants of the G guests (G¼AcOꢀ, Fꢀ, Clꢀ, HP2O37ꢀ
,
OHꢀ) to L, determined by UVevis, fluorescence, and NMR titration in DMSO/0.5%
water solution or CH3CN at 298 K
Reaction
log K
DMSO
AcOꢀ
CH3CN
AcOꢀ
Fꢀ
HP2O37ꢀ Fꢀ
Clꢀ
OHꢀ
LþG¼LG
4.3(2)a 2.7(1)a 7.8(1)b 6.4(1)b
6.5(2)b 5.6(1)b 5.6(1)b
6.3(3)c 5.5(3)c 5.6(1)c
5.2(1)b 4.5(1)b 4.7(1)b
5.1(2)c 4.3(2)c 4.6(1)c
7.5(1)c 6.6(1)c
LGþG¼LG2 2.8(1)a 1.3(1)a 5.6(1)b 3.8(1)b
5.3(1)c 4.4(1)c
a
Values obtained by NMR.
Values obtained by UVevis.
Values obtained by fluorescence experiments.
Values in parentheses are the standard deviation to the last significant figure.
Alternatively, reagents with two different leaving groups at-
tached to the carbonyl group, i.e., unsymmetrical phosgene de-
rivatives, could be utilized. Here, it is possible to control the rate of
b
c
d
Scheme 1. Synthesis of L.

3770
G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
2.3. NMR experiments
bonding interaction between L and the two anions. Similar exper-
iments carried out with HP2O37ꢀ showed an analogous behavior and
only a large excess of this anion deprotonates L, while it is bound
when present in an equimolar ratio.
The interaction of L with anions G (G¼AcOꢀ, Fꢀ, Clꢀ, Brꢀ, HP2O37ꢀ
and OHꢀ) were studied in DMSO-d6/0.5% water solution at 303 K.
Fig. 2 reports the 1H NMR screening of the interaction of L with such
anions. As with similar urea-based systems,24,25 the main changes
are the downfield shifts of the H4 and H5 ureido signals or their
disappearance. The former is due to the hosteguest recognition
processes occurring via H-bonding to the ureido moieties, the sec-
ond to a deprotonation reaction. Upon addition of the anion, with
the exception of Brꢀ, the L spectrum undergoes modifications due to
the hosteguest interaction with Clꢀ, Fꢀ, and AcOꢀ; while deproto-
nation with OHꢀ and HP2O37ꢀ causes spectral changes (Fig. 2). For
example, H5 and H4 are 1.95 and 1.47 ppm, respectively, downfield
shifted by adding AcOꢀ; no deprotonation process was observed
with this anion, as also confirmed by spectrophotometric mea-
surements. On the contrary, the spectrum recorded in a large excess
of Bu4NOH shows the disappearance of the ureido protons due to the
deprotonation process of such groups; this was also confirmed by
the upfield shift of the coumarin H6, H7, and H9 resonances, sug-
gesting that H5 is the more acidic proton of the group. The hoste-
guest recognition and the deprotonation processes affect the H6 and
H10 protons in the ortho position of the coumarin differently. Anion
binding gives rise to a shift of H10 (up to 0.15 ppm) as H6 remains
relatively unchanged, while deprotonation also shifts H6 (Fig. 2).
2.4. UVevis and fluorescence experiments
The UVevis absorption spectrum of L in both DMSO and CH3CN
undergoes modifications when anion G is bound. As an example,
Fig. 5a shows the titration spectra with acetate performed in CH3CN.
In the absence of anion, L shows a strong absorption band in the
263e380 nm range with the maximum absorption centered
at 328 nm ascribed to the coumarin-ureido moiety
(
3
¼26,300 cmꢀ1 molꢀ1 dm3). The addition of acetate, as the tetra-
butylammonium salt, leads to a shift of the band toward lower en-
ergies reaching, after the addition of about 4 equiv of AcOꢀ (see the
inset of Fig. 5a), the maximum shift lmax at 335 nm
(
3
¼33,300 cmꢀ1 molꢀ1 dm3). These spectral modificationshighlight,
as already observed in the 1H NMR experiments, acetate binding
with the formation of LeAcOꢀ adducts able to affect the chromo-
phore. As already reported, acetate does not induce deprotonation of
the ureido NH protons, even when a large excess of anion was used.
Comparable results with a similar spectral trend were obtained
with fluoride and chloride, while, as reported in the NMR results,
OHꢀ and HP2O37ꢀ exhibited different behavior giving rise to a pre-
Fig. 2. 1H NMR spectrum in DMSO of L (1ꢁ10ꢀ2 mol dmꢀ3) and of L after the addition of 5 equiv of Bu4NBr, Bu4NF, Bu4NCl, Bu4NOAc, (Bu4N)3HP2O7, and Bu4NOH.
Figs. 3 and 4 show that L interacts with the fluoride anion
without a deprotonation process; in fact, after the addition of two
Bu4NF equivalents, both ureido NH protons are still visible in the
spectrum. This indicates that the coumarineurea is not acidic
enough to be deprotonated by fluoride. This characteristic allows
the determination of addition constants with fluoride, while the
same is not possible with similar urea-based systems.25 With the
hydroxide anion, only a large excess deprotonates L: in contrast,
a hosteguest interaction can be detected when they are present in
an equimolar ratio (see Fig. 3).
liminary interaction with L and a subsequent deprotonation pro-
cess when they are present in a large excess. The lmax at the end of
the titration for Fꢀ and Clꢀ are 337 and 333 nm, respectively; the
titration with Fꢀ is also reported in Fig. 6a.
The deprotonation occurring with an excess of HP2O37ꢀ and OHꢀ
anions induced a red shift of the absorption band (lmax¼408 nm)
with
a
simultaneous increase in absorptivity
(
3 ¼41,300
cmꢀ1 molꢀ1 dm3) as reported in Fig. 7a. The presence of the cou-
marin unit makes L strongly fluorescent, which allows the possi-
bility of exploiting this technique in signaling the interaction with
anions. Free L in CH3CN exhibits an emission band with lem at
400 nm (lex¼328 nm); the addition of anions affects the emission
and titration experiments can be carried out to evaluate the sta-
bility constants.
Fig. 4 shows that the downfield shift of the urea protons of the
LeCl adduct (Dd 0.13 ppm for H4, 0.25 ppm for H5) are similar to
those observed for the addition of 1 equiv of OHꢀ
(Dd 0.14 ppm for
H4, 0.20 ppm for H5), thus suggesting the presence of a similar H-

G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
3771
Fig. 3. 1H NMR titration of the LeFꢀ system in DMSO-d6/0.5% water solution at 298 K obtained by adding several amounts of Bu4NF to a L (1ꢁ10ꢀ2 mol dmꢀ3) solution; inset:
chemical shift of the amide NH proton H5 of L upon increasing concentration of Bu4NF.
The addition of AcOꢀ, Clꢀ, and HP2O37ꢀ and OHꢀ in equimolar
ratios of L/G decreases the emission in a similar way, without
reaching complete fluorescence quenching, (see Fig. 5b for AcOꢀ);
on the contrary, the emission is strongly enhanced in the presence
of fluoride (Fig. 6b). The addition of fluoride induces an increase of
the fluorescence emission together with a red shift of the band up
to 412 nm. The spectrum obtained after the addition of 3 equiv of
fluoride is not further modified. The fluorescence increase is due to
the LeFꢀ hosteguest recognition because the emission band at
480 nm, due to the deprotonated receptor, was not observed
(Fig. 7b). Notably, the fluorescence is 2.5-fold enhanced, giving rise
to a favorable case of selective offeon fluorescence response to the
presence of this anion in solution; this means that fluoride is the
only one, with respect to the other halides as well as competitive
anions, to exhibit this behavior. As reported above, the presence of
a large excess of OHꢀ and HP2O37ꢀ induces a deprotonation process
giving rise to the appearance of a new emission band centered at
480 nm and 474 nm, respectively. The spectrum recorded in the
presence of excess Bu4NOH is reported in Fig. 7b together with that
of the free L. Similar results were obtained in DMSO with a small
change in the absorption and emission wavelengths. It is known
that the photochemical behavior of 7-aminocoumarin dyes is reg-
ulated by an equilibrium between the planar highly emitting in-
ternal charge transfer (ICT) and the pyramidal non-emitting
twisted intramolecular charge transfer (TICT) excited states.32 This
equilibrium depends on several factors, such as the polarity of the
solvent, the presence of intra- and intermolecular H-bonding and
the availability of the lone pair of electrons of the conjugated ni-
trogen atom. Furthermore, when coumarin is inserted in a ligand
such as L, other phenomena, for example, PET, could take place and
Fig. 4. 1H NMR spectrum of L DMSO-d6/0.5% water solution at 298 K (a) and L after the addition of 1 equiv of Bu4NCl (b) Bu4NOH (c) and the addition of 50 equiv of Bu4NOH (d);
[L]¼1ꢁ10ꢀ2 mol dmꢀ3
.

3772
G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
Fig. 5. UVevis [L]¼2.7ꢁ10ꢀ5 mol dmꢀ3 (a) and fluorescence titration [L]¼
3.8ꢁ10ꢀ6 mol dmꢀ3 (b) of L with AcOꢀ in CH3CN solution at 298 K. Inset: variation of
the labs at 335 nm (a) and lem at 400 nm (b) as a function of AcOꢀ added.
Fig. 6. UVevis [L]¼2.7ꢁ10ꢀ5 mol dmꢀ3 (a) and fluorescence titration [L]¼
3.8ꢁ10ꢀ6 mol dmꢀ3 (b) of L with Fꢀ in CH3CN solution at 298 K. Inset: variation of the
labs at 335 nm (a) and lem at 400 nm (b) as a function of Fꢀ added.
perturb the emission properties. The binding of fluoride probably
increases the population of the ICT with respect to the TICT state
giving rise to enhanced fluorescence; in fact, it is presumed that L
forms a stronger H-bond with Fꢀ than the other anions (NH/G)
making the N7 lone pair of electrons more available for conjugation
with the coumarin ring thus favoring the ICT state. On the contrary,
a weaker H-bond interaction in the chloride and oxygenate ad-
ducts, as well as a rigid conformation of LeAcO adducts (Scheme 2),
probably increases the flexible TICT despite the more rigid and
sterically hindered ICT state, causing fluorescence quenching. This
hypothesis is supported by the 1H NMR spectra in which the aro-
matic proton H10 shifts upfield in the fluoride adduct, while it shifts
downfield in the presence of the other interacting anions (Fig. 2).
In summary, the absorption and emission properties of L are
both affected, allowing the detection of the anion in solution. The
fluorescence behavior with the fluoride anion is the most in-
teresting. Fig. 8 shows the histograms obtained by plotting the
relative emission intensity (I/I0) of L in the presence of the various
anions investigated; while all of the other anions decrease the
emission, fluoride is the only one that enhances it, thus suggesting
the use of L as a selective fluorescence sensor for this anion. The
visible emission of L in the presence of several anions is reported in
Fig. 9, highlighting the change occurring in the presence of fluoride
to the naked eye.
NMR experiments. L is able to bind G (G¼AcOꢀ, Fꢀ, Cl, HP2O37ꢀ, and
OHꢀ) in both solvents forming adducts with 1:1 and 1:2 stoichi-
ometries. Acetate is the most tightly bound anion in both solvents;
in DMSO, the addition of the first acetate is more favored than the
second one (log K1¼4.3, log K2¼2.8) and it is also more favored with
respect to values found when only one ureido fragment stabilizes
the anion.33 This suggests that both ureido moieties of L cooperate
in the stabilization of the first AcOꢀ, as already found in amino-
based bis-ureido ligands,24,25 by forming a multiple H-bonding
network as schematically depicted in Scheme 2. The addition of the
second acetate to form the [L(AcO)2]2ꢀ species was less favored
when compared to the former for obvious steric hindrance reasons
as well as electrostatic repulsions.
As usually expected for these solvents,33 the values found in
CH3CN are higher than in DMSO although the same LeAcOꢀ spe-
ciation was preserved (log K1¼7.8, log K2¼5.6); thus a similar co-
ordination, at least for the first AcOꢀ, can also be hypothesized in
this medium. Hydrogenpyrophosphate had a first addition constant
value (log K1¼6.4) much higher than the second one (log K2¼3.8)
suggesting stabilization due to the first HP2O37ꢀ similar to that hy-
pothesized for the first acetate with a cooperative contribution of
both ureido groups. Halides showed the formation of the same
[LG]ꢀ and [LG2]2ꢀ adducts. Fluoride exhibited higher constant
values than chloride in both addition reactions. In addition, fluoride
showed higher addition constant values in CH3CN than in DMSO.
The addition of the second halide to form the [LG2]2ꢀ species is less
favored than the former. Considering that hydroxide deprotonates
L only when it is present in large excess, it was also possible to
2.5. Addition constants
Table 1 reports the stability constants obtained by UVevis and
fluorescence titration experiments in CH3CN and those obtained by
1H NMR in DMSO, since L was not soluble enough in CH3CN for the

G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
3773
Fig. 8. Histograms of the relative emission intensity (IꢀI0/I0) in CH3CN of L in the
presence of 5 equiv of HP2O37ꢀ, AcOꢀ, OHꢀ, Fꢀ, and Clꢀ anions.
Fig. 9. Photo of L in CH3CN in the presence of 5 equiv of AcOꢀ, HP2O37ꢀ, Fꢀ and 50 equiv
of OHꢀ
.
was obtained with a 65% overall yield in very mild synthetic con-
ditions by reacting triamine with the new activated 7-
1
aminocoumarin derivative 6. The anion coordination properties
highlighted that L is able to bind anions (G) AcOꢀ, Fꢀ, Clꢀ, OHꢀ, and
HP2O37ꢀ, although the two latter anions deprotonate L when they
are present in large excess in solution. The stability constants ac-
count for the formation of [LG] and [LG2] species with all inter-
acting anions. Acetate is the most tightly bound anion in both
speciations. The higher value of the first addition constant than the
second one for acetate and pyrophosphate suggests the co-
operation of both ureido units in stabilizing the first anion bound;
while this cannot be hypothesized for halides or hydroxide, which
show similar constant values for the first and second anion addi-
tions. L responds to the presence of a guest with optical and NMR
changes. Particularly interesting is the fluorescence response to
fluoride, which was the only anion that enhanced of the fluores-
cence in the visible range. In conclusion, the insertion of a coumarin
unit in the urea moieties preserves the H-bonding donor properties
of the latter toward H-bonding acceptor anions; the assembly of
two binding groups spaced by a flexible linear amine chain allows
the cooperation of both ureido binding units to bind multiple H-
bonding acceptor anions such as acetate and pyrophosphate, giving
more stable adducts; and the fluorescence response is shifted to-
ward visible wavelengths leading to a selective switch on response
to fluoride. This is desirable because for many applications it is
known that either thermodynamically interfering analytes are not
present in the analyzed matrix, or only a qualitative response to the
presence of the target species in the matrix is required. As a result,
current research in the field, aspires to obtain optical selectivity in
the design of new fluorescent chemosensors rather than striving
for thermodynamic selectivity. Studies to directly link the central
nitrogen atom of the new fluorosensor, through a suitable spacer in
place of the methyl group, to a polymeric framework are under
progress in our laboratory.
Fig. 7. UVevis spectra of L free and after the addition of 50 equiv of Bu4NOH, [L]¼
3.1ꢁ10ꢀ5 mol dmꢀ3 (a) fluorescence spectra (lex¼328 nm) of L free and after the ad-
dition of 50 equiv of Bu4NOH, [L]¼3.8ꢁ10ꢀ6 mol dmꢀ3 (b) in CH3CN.
Scheme 2. Proposed multiple H-bonding network for acetate.
evaluate the addition constants for hydroxide; the formation con-
stant values of [LOH]ꢀ and [L(OH)2]2ꢀ species are log K¼5.6 and 4.7,
respectively. As for halide guests, similar values between the first
and second anion additions suggest that these anions are bound
independently by the two ureido groups although a degree of co-
operation cannot be excluded.
3. Conclusions
A novel fluorescent anion receptor based on a bis-urea binding
moiety with coumarin as a signaling unit is reported. An efficient
one-pot two-step synthetic procedure was developed and ligand L

3774
G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
4. Experimental
(H3, 4H, m), 2.50 (H2, 4H, m), 2.34 (H8, 6H, d, J¼1.2 Hz), 2.26 (H1,
3H, s). 13C NMR (DMSO) 160.7, 155.2, 154.5, 153.6, 144.7, 126.1, 114.3,
113.5, 111.4, 104.0, 56.8, 42.2, 37.4, 18.4 ppm; MS (þESI) 520.2
(MHþ). Anal. for C27H29N5O6 (519.56): calcd C, 64.42; H, 5.63; N;
13.48; found C, 64.4; H, 5.6; N, 13.5.
4.1. General methods
IR spectra were recorded on a Shimadzu FTIR-8300 spectrom-
eter. Melting points were determined on a Buchi melting point B
540 apparatus and are uncorrected. EIMS spectra (70 eV) were
recorded with a Fisons Trio 1000 spectrometer. The HPLC system
(Waters Alliance 2795) was coupled with a photodiode array de-
tector (Waters 2996 PDA), followed by an electrospray mass spec-
trometer detector (ESI-MS) (Waters-Micromass ZQ) worked by
Mass Lynx 4.1 SP4 software. The ESI-MS analyses were performed
in a positive mode under the following conditions: source and
desolvation temperature 100 and 250 ꢂC; capillary and cone voltage
2.5 kV and 25 V; cone and desolvation flow (nitrogen gas) 40 and
400 L/h. HPLC analyses were performed on a 25 cmꢁ4.6 mm Dis-
covery C-18, 5 cm column (Supelco, Bellefonte, PA) equipped with
a Supelguard Discovery C-18 guard column (2 cmꢁ4 mm, 5 cm).
Solvents A (MeOH) and B (5 nM ammonium acetate) were run at
a flow rate of 1 mL/min. The running gradient was adjusted to 10% A
(2 min), increasing to 90% A (18 min), and then 100% C (2 min),
followed by a reequilibration at 10% A (15 min). All solvents were
HPLC-grade (AldricheSigma) and water was purified via a Millex Q-
plus system (Millipore).
4.2.3. Two-step one-pot procedure of L. To a well stirred solution of
7-amino-4-methylcoumarin 3 (0.81 g, 4.62 mmol) and pyridine
(0.392 mL, 4.85 mmol, 1.05 equiv) in dry CH2Cl2 (5 mL) was added
a solution of p-nitrophenyl chloroformate 5 (0.978 g, 4.83 mmol,
1.05 equiv) in CH2Cl2 (3 mL). The solution was stirred at room
temperature for 1 h, concentrated under reduced pressure. To
a stirring solution of 1 (0.50 g, 2.22 mmol) and DIPEA (2.4 mL) in
DMF (10 mL) was slowly added a solution of the above crude p-
nitrophenyl carbamate dissolved in DMF (5 mL). The homogeneous
solution was stirred for 3 h at room temperature, concentrated
under reduced pressure, and chromatographed (SiO2; DCM/MeOH
90:10 to DCM/MeOH/NH4OH 90/07/03; Rf¼0.23) to give 1.15 g (65
%) of L as a white solid.
4.3. Spectroscopic experiments
1H, 13C, and 19F NMR spectra were recorded on a Bruker Avance
200 instrument, operating at 200.13, 50.33, and 188.31 MHz, re-
spectively, and equipped with a variable temperature controller.
The temperature of the NMR probe was calibrated using 1,2-
ethandiol as calibration sample. For the spectra recorded in CDCl3,
CD3CN, and DMSO-d6 the 1H and 13C peak positions are reported
with respect to TMS while CFCl3 was used as standard reference for
the 19F NMR spectroscopy. 1He1H and 1He13C correlation experi-
All chemicals were purchased from Aldrich, Fluka, and Lancaster
in the highest quality commercially available. All the solvents were
dried prior to use. Chromatographic separations were performed
on silica gel columns by flash chromatography (Kieselgel 60,
0.040e0.063 mm, Merck). TLC analyses were performed on pre-
coated silica gel on aluminum sheets (Kieselgel 60 F254, Merck).
ments were performed to assign the signals. Chemical shifts (
d
4.2. Synthesis
scale) are reported in parts per million (ppm values) relative to the
characteristic peak of the solvent; coupling constants (J values) are
given in hertz (Hz).
4.2.1. 4-Nitrophenyl
N-(4-methyl-2-oxo-2H-chromen-7-yl)carba-
mate (6). To a well stirred solution of 7-amino-4-methylcoumarin
3 (1.00 g, 5.71 mmol) and pyridine (0.49 mL, 6.0 mmol, 1.05 equiv)
in dry CH2Cl2 (5 mL) was added a solution of p-nitrophenyl chlor-
oformate 5 (1.21 g, 6.0 mmol, 1.05 equiv) in CH2Cl2 (4 mL). The
resulting solution was stirred at room temperature for 1 h, con-
centrated by reduced pressure, and used in the further step without
any additional purification. An analytical sample was diluted with
CH2Cl2 (15 mL), washed with water, 5% aqueous HCl and 5% aque-
ous NaHCO3, and dried (MgSO4). After evaporation, purification by
column chromatography (SiO2; cyclohexane/AcOEt 50:50 to 35:65)
provided 6 (1.70 g, 88%) as a pale-yellow solid. The residue was
crystallized from CHCl3 to afford N-p-nitrophenyl carbamate 6 as
pale-yellow crystalline needles. Mp¼130e131 ꢂC. 1H NMR (DMSO):
11.0 (br s, 1H), 8.34, (t, J¼10 Hz, 1H), 8.10 (d, J¼10 Hz, 1H), 7.78e7.56
(m, 1H), 7.39 (d, J¼10.0 Hz, 1H), 6.92 (d, J¼10.0 Hz, 1H), 6.56 (dd,
J¼2.0, 10.0 Hz, 1H), 6.4 (d, J¼2.0 Hz, 1H), 5.89 (s, 1H), 2.29, (s, 3H).
13C NMR (DMSO): 164.3, 161.1,155.9, 154.1, 153.4, 126.6, 126.5, 116.2,
111.6,109.3, 107.9, 99.0, 18.4 ppm. FTIR (Nujol): 1766, 1616, 1593,
NMR titrations were carried out in a DMSO-d6/0.5% water
mixture; 0.5% of water was added to DMSO to avoid the not uni-
form absorption of water from the atmosphere by anhydrous DMSO
during the titration. In a typical experiment, a 5ꢁ10ꢀ2 mol dmꢀ3
solution of the anion was added in 0.1 equiv at a time to
a 1ꢁ10ꢀ2 mol dmꢀ3 solution of the ligand directly in the NMR tube;
a 5 min free period was carefully respected before measurement.
All the spectra measurements were done at a temperature of 298 K.
No effort was made to maintain constant the ionic strength in order
to avoid competition of other anions. The anions tested were added
as their tetrabutylammonium salts. The monitoring of the shift of
the signals in the ligand spectra (see Discussion) permitted evalu-
ation of the addition constants ligand-anion using the HYPNMR
computer program.34 The constants evaluated by NMR titration
were obtained processing all the shifting resonances together or as
separate single set; no significant changes in the values obtained in
the two different data treatments were observed.
Fluorescence spectra were recorded at 298 K with Varian Cary
Eclipse spectrofluorimeter. UVevis absorption spectra were
recorded at 298 K with a Varian Cary-100 spectrophotometer
equipped with a temperature control unit. The interaction of an-
ions with ligands L1 was studied in similar conditions of the NMR
titration experiments using CH3CN or DMSO/0.5% water mixture as
solvent; solution containing the anion G (Fꢀ, Clꢀ, HP2O37ꢀ, OHꢀ or
AcOꢀ) up to 5 equiv with respect to the ligand was added to the
solution containing L1 ([L1]¼1.5ꢁ105 M). At least three sets of
spectrophotometric titration curves for each G/L system were
performed. All sets of curves were treated either as single sets or
as separate entities, for each system; no significant variations
were found in the values of the determined constants. The
HYPERQUAD computer program was used to process the spectro-
photometric data.35
1525, 1461, 1376 cmꢀ1
.
4.2.2. 1,11-Bis(4-methylcoumarin-7-yl)-6-methyl-1,3,6,9,11-
pentaazaundeca-2,10-dione (L). To a stirring solution of triamine
hydrochloride 1 (0.60 g, 2.66 mmol) and N,N-diisopropylethyl-
amine (DIPEA) (3 mL) in DMF (12 mL) was added a solution of the
above carbamate 6 (1.99 g, 5.85 mmol) dissolved in DMF (2 mL).
The yellow resulting solution was stirred at room temperature for
3 h, concentrated to reduced pressure, and purified by column
chromatography (SiO2; DCM/MeOH 90:10 to DCM/MeOH/NH4OH
90/07/03; Rf¼0.23) provided L (1.08 g, 78%) as a white solid.
Mp¼214e216 ꢂC. 1H NMR (DMSO) 9.12 (H5, 2H, s), 7.56 (H7, 2H, d,
J¼8.7 Hz), 7.52 (H10, 2H, d, J¼2.0 Hz), 7.22 (H6, 2H, dd, J1¼8.7 Hz,
J2¼2.1 Hz), 6.31 (H4, 2H, t, J¼5.2 Hz), 6.13 (H9, 2H, d, J¼1.3 Hz), 3.23

G. Ambrosi et al. / Tetrahedron 68 (2012) 3768e3775
3775
Licchelli, M.; Monzani, E. Org. Biomol. Chem. 2005, 3, 1495e1500; (d) Gunn-
laugsson, T.; Kruger, P. E.; Jensen, P.; Tierney, J.; Ali, H. D. P.; Hussey, G. M. J. Org.
Chem. 2005, 70, 10875e10878; (e) Amendola, V.; Boiocchi, M.; Colasson, B.;
Fabbrizzi, L. Inorg. Chem. 2006, 45, 6138e6147; (f) Turner, D. R.; Paterson, M. J.;
Steed, J. W. J. Org. Chem. 2006, 71, 1598e1608; (g) Allevi, M.; Bonizzoni, M.;
Fabbrizzi, L. Chem.dEur. J. 2007, 13, 3787e3795; (h) Pescatori, L.; Arduini, A.;
Pochini, A.; Ugozzoli, F.; Secchi, A. Eur. J. Org. Chem. 2008, 109e120; (i) Calta-
girone, C.; Hiscock, J. R.; Hursthouse, M. B.; Light, M. E.; Gale, P. A. Chem.dEur. J.
2008, 14, 10236e10243; (j) Meshcheryakov, D.; Arnaud-Neu, F.; Bohmer, V.;
Bolte, M.; Cavalieri, J.; Hubscher-Bruder, V.; Thondorf, I.; Werner, S. Org. Biomol.
Chem. 2008, 6, 3244e3255; (k) Amendola, V.; Bergamaschi, G.; Boiocchi, M.;
Fabbrizzi, L.; Milani, M. Chem.dEur. J. 2010, 16, 4368e4380; (l) Amendola, V.;
Fabbrizzi, L.; Mosca, L. Chem. Soc. Rev. 2010, 39, 3889e3915.
References and notes
1. Kirk, K. L. Biochemistry of the Halogens and Inorganic Halides; Plenum: New York,
NY, 1991, pp 58e86.
2. Riggs, B. L. In Bone and Mineral Research, Annual 2; Elsevier: Amsterdam, 1983,
pp 366e393.
3. Kleerekoper, M. Endocrinol. Metab. Clin. North Am. 1998, 27, 441e452.
4. Wiseman, A. In Handbook of Experimental Pharmacology XX/2; Springer: Berlin,
1990, pp 48e97, Part 2.
5. Weatherall, J. A. In Handbook of Experimental Pharmacology XX/1; Springer:
Berlin, 1969, pp 141e172, Part 1.
6. Kim, S. Y.; Hong, J.-I. Org. Lett. 2007, 9, 3109e3112.
7. Gunnlaugsson, T.; Davis, A. P.; O’Brien, J. E.; Glynn, M. Org. Lett. 2002, 4,
2449e2452.
8. (a) Abeysekara, M. S.; Naylor, J. M.; Wassef, A. W. A.; Isak, U.; Zello, G. A. Am J.
Physiol. Endocrinol. Metab. 2007, 293, 558e565; (b) Maus, M.; Marin, P.; Israel,
M.; Glowinski, J.; Premont, J. Eur. J. Neurosci. 1999, 11, 3215e3224; (c) Walsh, C.
Nature 2000, 406, 775e781.
9. Michigami, Y.; Kuroda, Y.; Ueda, K.; Yamamoto, Y. Anal. Chim. Acta 1993, 274,
299e302.
21. (a) Sessler, J. L.; Davis, J. M. Acc. Chem. Res. 2001, 34, 989e997; (b) Camiolo, S.;
Gale, P. A.; Hursthouse, M. B.; Light, M. E.; Shi, A. J. Chem. Commun. 2002,
758e759; (c) Camiolo, S.; Gale, P. A.; Hursthouse, M. B.; Light, M. E. Org. Biomol.
Chem. 2003, 1, 741e744; (d) Sessler, J. L.; Camiolo, S.; Gale, P. A. Coord. Chem. Rev.
2003, 240, 17e55; (e) Sessler, J. L.; Katayev, E.; Pantos, G. D.; Scherbakov, P.;
Reshetova, M. D.; Khrustalev, V.; Lynch, V. M.; Ustynyuk, Y. A. J. Am. Chem. Soc.
2005, 127, 11442e11446.
22. de Silva, A. P.; Guranatne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C.
P.; Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515e1566.
23. Rambo, B. M.; Silver, E. S.; Bielawski, C. W.; Sessler, J. L. Top. Heterocycl. Chem.
10. Ayoob, S.; Gupta, A. K. Crit. Rev. Environ. Sci. Technol. 2006, 36, 433e487.
11. Bassin, E. B.; Wypij, D.; Davis, R. B. Cancer Causes Control 2006, 17, 421e428.
12. Wang, S. X.; Wang, Z. H.; Cheng, X. T.; Li, J.; Sang, Z. P.; Zhang, X. D.; Han, L. L.;
Qiao, X. Y.; Wu, Z. M.; Wang, Z. Q. Environ. Health Perspect. 2006, 115, 643e647.
13. Yu, Y.; Yang, W.; Dong, Z.; Wan, C.; Zhang, J.; Liu, J.; Xiao, K.; Huang, Y.; Lu, B.
Fluoride 2008, 41, 134e138.
2010, 24, 1e37 and references therein.
24. Formica, M.; Fusi, V.; Macedi, E.; Paoli, P.; Piersanti, G.; Rossi, P.; Zappia, G.;
Orlando, P. New J. Chem. 2008, 32, 1204e1214.
25. Formica, M.; Fusi, V.; Giorgi, L.; Macedi, E.; Piersanti, G.; Varrese, M. A.; Zappia,
14. (a) Recognition of Anions, Structure and Bonding; Vilar, R., Ed.; Springer-Verlag:
G. Supramol. Chem. 2010, 22, 365e379.
Berlin, 2008; Vol. 129; (b) Supramolecular Chemistry of Anions; Bianchi, A.,
Bowman-James, K., Garcia-Espana, E., Eds.; Wiley-VCH: New York, NY, 1997;
pp 461e482; (c) Sessler, J. L.; Gale, P. A.; Cho, W. S. Anion Receptor Chemistry;
RSC: Cambridge, UK, 2006, pp 259e293.
26. For reviews on substituted ureas, see: (a) Gallou, I. Org. Prep. Proced. Int. 2007,
39, 355e383; (b) Bigi, F.; Maggi, R.; Sartori, G. Green Chem. 2000, 2, 140e148;
Vishnyakova, T. P.; Golubeva, I. A.; Glebova, E. V. Russ. Chem. Rev. (Engl. Ed.)
1985, 54, 249e261; For recent papers on the preparation of unsymmetrical
ureas: (c) Bogolubsky, A. V.; Ryabukhin, S. V.; Pipko, S. E.; Lukin, O.; Shiva-
nyuk, A.; Mykytenko, D.; Tolmachev, A. Tetrahedron 2011, 67, 3619e3623; (d)
Breitler, S.; Oldenhuis, N. J.; Fors, B.; Buchwald, S. L. Org. Lett. 2011, 13,
3262e3265.
27. Selected examples on substituted ureas as intermediates in organic synthesis,
see: (a) Gao, J.; Li, H.; Zhang, Y.; Zhang, Y. Green Chem. 2007, 9, 572e576; (b)
Clayden, J.; Hennecke, U. Org. Lett. 2008, 10, 3567e3570; (c) Clayden, J.; Dufour,
J.; Grainger, D. M.; Helliwell, M. J. Am. Chem. Soc. 2007, 129, 7488e7489; (d)
McCooey, S. H.; Connon, S. J. Angew. Chem., Int. Ed. 2005, 44, 6367e6370; (e) Yu,
S.; Haight, A.; Kotecki, B.; Wang, L.; Lukin, K.; Hill, D. R. J. Org. Chem. 2009, 74,
9539e9542.
~
15. (a) Caltagirone, C.; Gale, P. A. Chem. Soc. Rev. 2009, 38, 520e563; (b) Gale, P. A.
Acc. Chem. Res. 2006, 39, 465e475; (c) Suksai, C.; Tuntulani, T. Chem. Soc. Rev.
2003, 32, 192e202; (d) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40,
486e516; (e) Schmidtchen, F. P.; Berger, M. Chem. Rev. 1997, 97, 1609e1646.
16. (a) Gale, P. A. Coord. Chem. Rev. 2003, 240, 191e221; (b) Katayev, E. A.; Ustynyuk,
Y. A.; Sessler, J. L. Coord. Chem. Rev. 2006, 250, 3004e3037; (c) Lankshear, M. D.;
Beer, P. D. Coord. Chem. Rev. 2006, 250, 3142e3160; (d) Schmidtchen, F. P. Coord.
Chem. Rev. 2006, 250, 2918e2928; (e) Anzenbacher, P.; Nishiyabu, R.; Palacios,
M. A. Coord. Chem. Rev. 2006, 250, 2929e2938.
17. (a) Amendola, V.; Bonizzoni, M.; Esteban-Gomez, D.; Fabbrizzi, L.; Licchelli, M.;
Sancenon, F.; Taglietti, A. Coord. Chem. Rev. 2006, 250, 1451e1470; (b) Garcia
~
Espana, E.; Diaz, P.; Llinares, J. M.; Bianchi, A. Coord. Chem. Rev. 2006, 250,
28. For a review on urea-based bifunctional organocatalysts in organic synthesis,
see: Zhang, Z.; Schreiner, P. R. Chem. Soc. Rev. 2009, 38, 1187e1198 and refer-
ences therein.
2952e2986; (c) Kim, S. K.; Lee, D. H.; Hong, J.; Yoon, J. Acc. Chem. Res. 2009, 42,
23e31; (d) Cametti, M.; Rissanen, K. Chem. Commun. 2009, 2809e2829; (e)
Amendola, V.; Fabbrizzi, L. Chem. Commun. 2009, 513e531; (f) Gamez, P.;
Mooibroek, T. J.; Teat, S. J.; Reedijk, J. Acc. Chem. Res. 2007, 40, 435e444.
18. (a) Miyaji, H.; Sessler, J. L. Angew. Chem., Int. Ed. 2001, 40, 154e157; (b) Han, M.
S.; Kim, D. H. Angew. Chem., Int. Ed. 2002, 41, 3809e3811; (c) Martinez-Manez,
R.; Sancenon, F. Chem. Rev. 2003, 103, 4419e4476; (d) Jose, D. A.; Kumar, D. K.;
Ganguly, B.; Das, A. Org. Lett. 2004, 6, 3445e3448.
19. (a) Coles, S. J.; Frey, J. G.; Gale, P. A.; Hursthouse, M. B.; Light, M. E.; Navakhun,
K.; Thomas, G. L. Chem. Commun. 2003, 568e589; (b) Bondy, C. R.; Loeb, S. J.
Coord. Chem. Rev. 2003, 240, 77e99; (c) Brooks, S. J.; Evans, L. S.; Gale, P. A.;
Hursthouse, M. B.; Light, M. E. Chem. Commun. 2005, 734e736; (d) Kang, S. O.;
Powell, D.; Day, V. W.; Bowman-James, K. Angew. Chem., Int. Ed. 2006, 45,
1921e1925; (e) Beer, P. D.; Sambrook, M. R.; Curiel, D. Chem. Commun. 2006,
2105e2117; (f) Garcia-Garrido, S. E.; Caltagirone, C.; Gale, P. A.; Light, M. E.
Chem. Commun. 2007, 1450e1452; (g) Korendovych, I. V.; Cho, M.; Makhlynets,
O. V.; Butler, P. L.; Staples, R. J.; Rybak-Akimova, E. V. J. Org. Chem. 2008, 73,
4771e4782; (h) Kang, S. O.; Day, V. W.; Bowman-James, K. Org. Lett. 2009, 11,
3654e3657.
29. (a) Babad, H.; Zeiler, A. G. Chem. Rev. 1973, 73, 75e91; Smith, M. B.; March, J.
March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th
ed;; Wiley: New York, NY, 2001; p 1191; (b) Knolker, H.-J.; Braxmeier, T.;
Schlechtingen, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 2497e2500.
30. The reaction of isocyanates with various nucleophiles has been extensively
reported in the literature, see: Braunstein, P.; Nobel, D. Chem. Rev. 1989, 89,
1927e1945; For recent examples for the preparation of ureas via in situ for-
mation of isocyanate, see: (a) Lebel, H.; Leogane, O. Org. Lett. 2006, 8,
ꢀ
5717e5720; (b) Dube, P.; Noah, F.; Nathel, F.; Vetelino, M.; Couturier, M.;
Aboussafy, C. L.; Pichette, S.; Jorgensen, M. L.; Hardink, M. Org. Lett. 2009, 11,
5622e5625; (c) Peterson, S. L.; Stucka, S. M.; Dinsmore, C. J. Org. Lett. 2010, 12,
1340e1343.
31. Sammet, B. Synlett 2009, 3050e3051.
32. Satpati, A. K.; Kumbhakar, M.; Nath, S.; Pal, H. Photochem. Photobiol. 2009, 85,
119e129 and references therein.
ꢀ
33. Perez-Casa, C.; Yatsimirsky, A. K. J. Org. Chem. 2008, 73, 2275e2284.
34. Frassineti, C.; Ghelli, S.; Gans, P.; Sabatini, A.; Moruzzi, M. S.; Vacca, A. Anal.
Biochem. 1995, 231, 374e382.
35. Gans, P.; Sabatini, A.; Vacca, A. Talanta 1996, 43, 1739e1753.
20. (a) Sansone, F.; Chierici, E.; Casnati, A.; Ungaro, R. Org. Biomol. Chem. 2003, 1,
1802e1809; (b) Gunnlaugsson, T.; Davis, A. P.; Hussey, G. M.; Tierney, J.; Glynn,
M. Org. Biomol. Chem. 2004, 2, 1856e1863; (c) Gomez, D. E.; Fabbrizzi, L.;