Pd cross-coupling reactions in the access to bis-pyrazole and bis-indazole pyridine-based nona-coordinated ligands. Luminescence properties of their lanthanide complexes

Article abstract of DOI:10.1016/j.jphotochem.2017.03.037

A synthetic approach based on microwave-assisted Pd cross-coupling reactions is developed to obtain two series of push-pull donor-π-conjugated nonadentate coordinating ligands constructed on bispyrazolylpyridine and bisindazolylpyridine units. Both famili

Full text of DOI:10.1016/j.jphotochem.2017.03.037

Accepted Manuscript
Title: Pd cross-coupling reactions in the access to bis-pyrazole
and bis-indazole pyridine-based nona-coordinated ligands.
Luminescence properties of their lanthanide complexes
Authors: Arturo Gamonal, Ernesto Brunet, Olga Juanes, Juan
Carlos Rodriguez-Ubis
PII:
S1010-6030(17)30221-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.03.037
JPC 10588
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
20-2-2017
27-3-2017
30-3-2017
Please cite this article as: Arturo Gamonal, Ernesto Brunet, Olga Juanes, Juan
Carlos Rodriguez-Ubis, Pd cross-coupling reactions in the access to bis-pyrazole
and bis-indazole pyridine-based nona-coordinated ligands.Luminescence properties
of their lanthanide complexes, Journal of Photochemistry and Photobiology A:
Chemistryhttp://dx.doi.org/10.1016/j.jphotochem.2017.03.037
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Graphical abstract
1

Highlights
A versatile synthetic approach for best and most effective markers for bioassays is
developed.
This straightforward synthetic access to 4-substituted pyridines, allows easy
optimization of the emission properties of lanthanides.
The synthesis is based on microwave-assisted Pd cross-coupling reactions, to
obtain photoactive push-pull donor-π-conjugated ligands.
The ligands constructed on bispyrazolylpyridine and bisindazolylpyridine units
formed stable complexes with Europium and Terbium in H2O
In an effort to shed light on the nature of the structure-property relationships, a
study of the effect of different substituents on their photophysical behavior is
presented.
2

Pd cross-coupling reactions in the access to bis-
pyrazole and bis-indazole pyridine-based nona-
coordinated ligands. Luminescence properties of
their lanthanide complexes
Arturo Gamonal, Ernesto Brunet, Olga Juanes and Juan Carlos Rodriguez-Ubis*1.
Address: 1 Departamento de Química Orgánica, Facultad de Ciencias, Universidad
Autónoma de Madrid, 28049-Madrid, Spain
Email: jcrubis@uam.es
* Corresponding author
Abstract
A synthetic approach based on microwave-assisted Pd cross-coupling reactions is
developed to obtain two series of push-pull donor-π-conjugated nonadentate
coordinating ligands constructed on bispyrazolylpyridine and bisindazolylpyridine
units. Both families of ligands formed stable complexes with Europium and Terbium
in H2O. To provide a basis to synthesize the best and most effective markers for
bioassays, a study of the effect of different substituents on their photophysical
behavior is presented in an effort to shed light on the nature of the structure-property
relationships. Both families showed better emission properties for Tb(III) than for
Eu(III). Bispyrazolylpyridine derivatives were revealed as better sensitizing ligands
despite the promising higher absorptivity and red-shifted excitation wavelengths of
their counterpart, bisindazolilpyridine. This straightforward synthetic access to 4-
substituted pyridines allows easy optimization of the emission properties of
lanthanides. In the case of 4-phenylethynyl substituted bispyrazolylpyridine, the
3

emission properties of Eu(III) and Tb(III) complexes were similar, indicating that they
can act as a promising long-lived luminescent dual probe in multiplex analysis.
Keywords
Energy transfer; Inner-sphere hydration number; Lanthanide(III) ions; Luminescence
lifetime; Polyaminopolycarboxylate complexes
1. Introduction
The luminescence emissions exhibited by lanthanide (III) complexes with organic
chromophoric ligands have stimulated great interest in being indebted to the wide-
ranging applicability of their narrow-band emissions as probes in a variety of
biological applications [1], cell and tissue imaging [2] and Ln-based nanomaterials
[3]. The attractive features of lanthanide ions when they are complexed with
chromophoric organic ligands, are principally governed by their sensitivity and
selectivity, attained by their line-like emission, large Stokes shift, high quantum yield,
long luminescence lifetime and high photochemical stability [4, 5].
Since the first tetracarboxylic podands were published by our research group in 1993
[6], there have been many efforts to synthesize new nona-coordinated ligands due to
the good luminescence properties shown by their complexes with lanthanide ions [7-
9], making them excellent tools in homogeneous bioassays [10,11].
These ligands are constituted by a chromophore frame based on bispyrazolylpyridine
system, and two iminodiacetic acid units, which act as complexing agents. The cavity
created is able to host effectively the lanthanide ion isolating its coordination sphere
from the surrounding interactions. As a result of the effectiveness in coordination
these ligands exhibit good quantum yields for their complexes with Eu (III) and
especially for those of Tb (III). Other approaches to this type of structure have been
4

synthesized by our research group where different modifications of the chromophore
or complexing units were introduced to study their influence [12,13].
However, the synthetic approach to obtaining systematic variation on the pyridine
ring on the polyheterocyclic chromophore presented some limitations; pyrazoles were
introduced by nucleophilic substitution of 2,6-dibromopyridines in harsh conditions
and these bromines limit the number of possible functions that can be introduced in
the pyridine ring.
Recently the easy substitution of fluorine atoms developed by Slosscher [14,15] has
been employed by other groups to widen the synthesis for these type of compounds
by means of 4-bromo-2,6-difluoropyridine [9,16]. Taking advantage of the favorable
reaction conditions offered by this trihalogenated molecule for the orthogonal access
to trisubstituted pyridines, herein we report the synthesis of two families of tris-
heterocyclic ligands based on 2,6-bis(pyrazol-1-yl)pyridine (1-bpp) and 2,6-
bis(indazol-1-yl)pyridine (1-bip) with different substitution at the C4 position of the
pyridine ring obtained by means of Pd cross-coupling reactions (Scheme 1). The
possibility of introducing almost unlimited aromatic substituents on the 4 position of
the pyridine by means of a universal reaction is an interesting synthetic challenge
that could help to tune the sensitization properties of the chromophoric unit,
enhancing the luminescent emission of lanthanide complexes with these ligands.
5

Scheme 1: Ligands described in this work
Furthermore, to extend these ideas, indazolyl groups are newcomers to these
families of antenna ligands and we consider it interesting to compare both
heterocycles, pyrazole and indazole as sensitizers and chelating points of lanthanide
ions in a series of compounds described here, in which we can compare the effect of
these rings with same aromatic substituents at C-4 introduced by Suzuki-Miyaura and
Sonogashira reactions. These substituents are an example of the variety of push-pull
donor-π-conjugated aromatic groups that could be introduced to these systems. They
could be extended in the future to a wide variety of systems for tuning the
sensitization capabilities toward other lanthanides in an effort to obtain new chelates
for the simultaneous detection of multiple analytes; therefore, enhancing the efficacy
of bioassays by conducting just a single multiplexed bioanalysis.
2. Synthesis of Ligands
Starting from the 4-Bromo-2,6-difluoropyridine, disubstituted pyridines 1a-b were
obtained by nucleophilic substitution of the fluorine atoms with the sodium salts of 3-
ethoxycarbonylpyrazole or 3-methoxycarbonylindazole obtained by the treatment of
the corresponding esters with sodium hydride (Scheme 2). The reaction requires mild
conditions for pyrazole substitution where the reaction completes in 8h at room
temperature in THF, whereas for indazole substitution, the reaction requires 50ºC for
4h in DMF as a solvent in a similar manner, as described in [16]. The introduction of
the first indazole ring deactivates the second fluorine atom substitution, requiring
more severe conditions. In our hands we obtained the monosubstituted 2-(3-
methoxycarbonylindazol-1-yl)-4-bromo-6-fluoropyridine as the only product for the
reaction between indazole and pyridine in THF at room temperature; this
6

monosubstituted product has been employed for access to new families of
unsymmetrically substituted pyridines used as antenna ligands in the bioanalysis of
DNA [17].
The brominated position was, at this stage, employed for the introduction of different
aromatic groups by Pd coupling reactions. Two kinds of reactions were performed, as
described in the Scheme 2; Suzuki-Miyaura coupling with phenyl, thienyl and 4-
methoxyphenyl boronic acids and Sonogashira coupling with ethynylbenzene. Both
reactions were optimized using microwave-assisted conditions, which drastically
reduce the reaction times without affecting the reaction yields compared to
conventional methods; this methodology has been extended to other ethynyl
derivatives and boronic acids by our group [17].
R2
R2
Br
Br
N
i)
ii)
iii)
N
N
N
N
N
N
N
N
N
N
N
N
N
N
F
F
N
RO2C
CO2R
RO2C
CO2R
1a R=Et
1b R=Me
2aa-ad
2ba-bd
3aa-ad
OH 3ba-bd
HO
R2
R2
R2
R2
v)
vi)
N
N
N
N
iv)
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
OH
HO
Br
Br
N
N
N
N
CO2tBu
tBuO2C
3aa-ad
3ba-bd
4aa-ad
4ba-bd
CO2H
HO2C
CO2tBu
5aa-ad
5ba-bd
CO2H
tBuO2C
HO2C
Lpz a-d
Lind a-d
Scheme 2: i) 2 equiv ethoxycarbonylpyrazole, NaH (2.2 equiv), THF, r.t., 4h for 1a, or
2 equiv methoxycarbonylindazole, NaH (2.2 equiv), DMF, 40ºC, 5h for 1b ii) R2-
B(OH)2, THF, KOH, Pd(PPh3)4, 130ºC MW or ethynylbenzene, THF, Et3N,
PdCl2(PPh3)2, CuI, MW, 125ºC. iii) LiAlH4, THF. iv) PBr3, CH3CN; v)
t
NH(CH2CO2 Bu)2, CH3CN; vi) TFA/ CH2Cl2.
7

Transformation of esters 2aa-d and 2ba-d to the tetracarboxyloic ligands (Lpz-inda-d)
was accomplished as depicted in Scheme 2, following the procedures previously
reported by us [6,12,18].
3. Results and Discussion
Lanthanide complexes of the tetraacids Lpza-d and Linda-d were prepared by mixing
equimolecular amounts of ligand and lanthanide salts (LnCl3·6H2O) in borate buffer
(pH = 8.1). Tables 1-3 summarize the main photophysical properties of the
complexes obtained.
3.1 UV-Vis absorption spectra
The UV-Vis absorption spectra of ligands show a similar appearance with same
substituent at position C-4 of the pyridine, in both families of compounds, 1-bpp and
1-bip. Absorption maxima are red shifted by about 10-30 nm for 1-bip related to 1-
bpp as a consequence of the more conjugated indazole ring and planar disposition of
the bis-indazolylpyridine heterocyclic system. This value is especially interesting as
less energetic excitation maxima could drive to a better match of the excited states of
ligand and resonance energy level of lanthanide, mainly in Eu (III). In addition, lower
excitation energy is also interesting as it reduces the cost of quartz materials
associated with UV range measurements.
The absorption maxima for the complexes in 1-bpp derivatives Lpza-d show a
bathochromic shift of the absorption maxima related to the free ligands, whereas this
bathochromic effect is less pronounced for the ligands 1-bip Linda-d. Assuming that
the relative disposition of the three heterocyclic rings around the lanthanide ion must
be very similar in the complexes of both families, the different bathochromic shift
found probably has its origin in the more coplanar original disposition of the bis-
indazole ligands compared with their bis-pyrazole equivalents.
8

3.2 Lanthanide Emission Spectra
In all complexes, excitation into the longest absorption maxima wavelength results in
the observation of the distinctive emission patterns of the Eu(III) or Tb(III) ions
(Figure 1). The excitation spectra were in perfect agreement with the absorption
observed.
Figure 1: UV/Vis absorption spectrum of free ligand Lpza (solid black line),
excitation/emission (λ em=616 nm, λ exc=320 nm) spectra of Eu-Lpza (red line) and
excitation/emission (λ em=545 nm, λ exc=320 nm) spectra of Tb-Lpza (green line) in
borate buffer.
Observed emission intensities are generally stronger for the Tb(III) complexes
compared with their Eu(III) counterpart. The quantum yield described by Yuan’s
group for Tb-Lpza (ϕ =1) [7] is confirmed in our hands. The excellent availability of this
compound by the synthetic pathway herein described gives a tremendous
opportunity for the application of this ligand as a luminescent probe.
9

Another ligand whose synthesis has also been described by Yuan's group was
thienyl derivative Lpzd which presents with Eu(III) a quantum yield in our hands, of
only 0.02 slightly lower than previously described [19], and the only ligand where the
Eu(III) emission is higher than for Tb(III). Despite the good sensitization properties of
this thienyl aromatic heterocycle described for lanthanide complexes of
thenoyltrifluoroacetone, our thienyl ligands Lpzd and Lindd present low emission
intensities with the bisindazole compound being the lowest in the series.
The lower emission of the Linda-d complexes is a constant in all of the products
described here, which shows that despite their promising lower-energy excitation
band, these ligands are not as good sensitizers as the corresponding bispyrazolyl
Lpza-d. A similar behavior was observed by Charbonniere et al. for the only example
of these type of 1-bisindazole pyridine described until now [16].
Analyzing the measured lifetimes for these complexes, presented in Table 1, we can
obtain some explanations about the emissions observed. The lifetimes measured at
room temperature show the normal behavior observed for Eu(III) and Tb(III)
complexes where longer values correspond to the Tb(III) complexes. The values
increase when the lifetimes were measured in D2O at room temperature and at 77K.
The lifetime dependence on temperature evinced the deactivation by thermal
coupling, both radiant and non-radiant, which is non-negligible.
Table 1: Absorptivity (ε) and Lifetime data in H2O and D2O at rt and 77 K (ms).
10

ε [103·M-1·cm-1]
(λmax [nm])a
b
b
b
b
I
ems*ε
τH2O τD2O τD2O (77k)
τH2O τD2O τD2O (77k)
Tb
Eu
(λmax [nm])a
[ms]
[ms]
[ms]
3.68
3.22
2.88
2.31
2.82
1.87
2.74
2.75
[ms]
[ms]
[ms]
Lpza 11.5 (320) 2.68 2.92
Lpzb 24.5 (306) 1.66 2.29
Lpzc 19.7 (312) 2.43 2.59
Lpzd 32.9 (317) 1.93 2.3
LPza 11.3 (320) 1.35 2.63
Lpzb 25.1 (306) 1.38 2.26
Lpzc 19.1 (312) 1.35 2.19
LPzd 34.5 (317) 1.32 2.31
Linda 9.2 (323) 1.41 2.03
Lindb 25.1 (329) 1.38 1.81
Lindc 20.4(338) 1.16 1.93
Lindd 28.1 (321) 0.8 1.64
3.77
2.52
2.29
2.66
2.36
1.95
2.21
1.99
Linda
9.4 (323)
1.01 1.48
Lindb 26.1 (329) 1.45 1.86
Lindc 17.9 (338) 1.02 1.47
Lindd 26.7 (321) 1.52 1.87
a Excitation λmax, b Measured in 0.05M NaHCO3-D2O buffer
From these values it is possible to also extract the number of coordinated water
molecules around the first coordination sphere of the Ln(III) ion. These values have
been calculated according to the three main methods described in literature, those of
Horrocks [20,21], Parker [22] and Kimura [23], and are presented in Table 2. These
methods take into account different deactivation parameters, but in most cases all
show similar values. In our case Horrocks’ and Parker’s values show very similar
records, with less than one coordinated water molecule, except for Linda and Lindc
which show two anomalously higher values with more than one water molecule. The
similar coordinating geometry around the lanthanide for all of the ligands described
suggests that these values are artifacts with a distinct origin than simple deactivation
by O-H thermal coupling. Kimura’s equation takes into account only lifetimes in water
at r.t.; these values are of special interest when comparing ligands with very similar
geometries, as is our case, as deactivation by temperature or vibronic coupling with
O-H oscillators must be very similar in all cases. The values obtained from Kimura’s
equations show large discrepancies with the other two methods, mainly for the Tb(III)
complexes, where in most cases, values much higher than for one water molecule
11

were found. Only Lpza and Lpzc show lower values more coherent with the other
methods.
Table 2: Number of water molecules calculated by indicated method
Tb
Horrocksa Parkerb Kimurac
Eu
Horrocksa Parkerb Kimurac
Lpza
Lpzb
Lpzc
Lpzd
Linda
Lindb
Lindc
Lindd
0.1
0.7
0.1
0.4
1.3
0.6
1.3
0.5
0.1
0.8
0.1
0.4
1.5
0.7
1.4
0.6
0.6
1.6
0.8
1.2
3.1
1.9
3.1
1.8
Lpza
Lpzb
Lpzc
Lpzd
Linda
Lindb
Lindc
Lindc
0.4
0.3
0.3
0.3
0.2
0.2
0.4
0.7
0.2
0.1
0.1
0.1
0.0
0.0
0.2
0.5
0.3
0.3
0.3
0.4
0.3
0.3
0.5
0.9
a According to Ref. [20]; b According to Ref. [22]; c According to Ref. [23]
These results provide evidence that deactivation could also have its origin in other
processes. Among them and mainly for the Tb(III) complexes a back-transfer process
is a common cause of deactivation. This is produced when the ligand excited state is
next in energy with the 5D4 resonance emissive level of Tb. In order to study this
back-transfer process, the energy measurement of the triplet excited state was
determined from the phosphorescence spectra of the Gd(III) complexes at 77K,
whose values are presented in Table 3.
Again we can observe a dependence of the lifetime values for the Tb(III) complexes
on the triplet state energy levels; once again the best emissive complexes with Lpza
and Lpzc showing the higher values of 3E00 (23365 and 23474cm-1) and τH2O (2.68
and 2.43ms). These energy levels are situated well above the 5D4 emissive levels of
Tb and an efficient energy transfer takes place as revealed by their quantum yields.
Other interesting examples are those of Lpzb and Lindc whose 3E00 level although
12

lower (22075 and 22124cm-1) also show high quantum yields (62 and 32%); their low
lifetimes at room temperature in water is recovered in D2O and also at 77K showing a
more sensitive behavior towards OH coupling vibrations and temperature.
The remaining 3E00 levels are well below 22000cm-1; these energy values are
recognized as the limits when the energy of the lowest triplet state of the ligand is at
the borderline for competitive back-transfer energy process [24]. Four ligands, Lpzd,
Linda, Lindb and Lindd present values under this limits and their quantum yields suffer
from this competitive process.
Table 3: Photophysical data of LnL in buffer borate
3E 00
ф TbL[%]
100
ф EuL [%]a
Lpza
Lpzb
Lpzc
Lpzd
Linda
Lindb
Lindc
Lindd
23365
22075
23474
20619
21413
21322
22124
21231
10
9
17
2
1
1
62
19
0.8
24
3
32
0.6
4
0.1
a Quantum yields were determined using Cs3[Eu(dpa)3] ( (Φ = 0.24) as a standard
[25].
For the Eu(III) complexes the number of coordinated water molecules is always less
than one and is very similar independent of the equation employed in the calculation,
which corroborated the good isolation of the lanthanide ion and supports the
deactivation process of their equivalent Tb(III) complexes as an explanation for the
anomalous q values observed for this ion.
13

The 3E00 energy range contains the europium emissive levels 5Di and the Eu(III)
luminescence deactivation is mainly governed by non-radiative transitions. The
strong isotopic variation of lifetimes shows the coupling with O–H vibronic oscillators
as the main deactivation route with temperature being less important. From the
luminescence lifetimes at rt and 77K and in H2O and D2O by means of the equations
stated by Sabattini et al [26], the values of radiative (kr) and non-radiative (knr) rate
constants, as well as ligand–metal energy transfer efficiency (η) and lanthanide
quantum yield (фM) were estimated (the most relevant ones are collected in the
supporting information file).
The variation of the 3E00 energy values, attending to the nature of substituents a-d in
both series Lpz and Lind show more variation in the case of 1-bpp than for the 1-bip
coordinating units. In the case of the 1-bpp unit, phenyl (a) and phenylethynyl (c)
substituents, 23365 and 23474cm-1 respectively, show higher similar energies
whereas electro-donating substituents (b) and (d) decrease the energy of the triplet
state; a small reduction for p-methoxyphenyl (b) (22075cm-1) and a very low value of
20619cm-1 for thienyl substituent (d) is observed. However, values with small
variation <900 cm-1 (22124-21231cm-1) are observed in Lind series. This small range
for the 1-bip triplet levels suggests that they are centered on the 1-bip frame with low
influence on the electron demanding nature of the substituent at position 4 of the
pyridine ring.
4. Conclusion
In summary the synthesis proposed opens the way to access an enormous variation
of trisubstituted pyridine ligands by microwave assisted cross-coupling reactions in
an easy and straightforward way.
14

From both families of compounds reported, the luminescent properties of the ligands
based on the 2,6-bispyrazolyl-1-pyridine pattern are quite sensitive to pyridine
substitution, whereas its counterpart 2,6-bisindazolyl-1-pyridine ligands show more
uniform triplet state levels, with these levels being centered in the tris-heterocyclic
motif. Despite the expectations generated by the introduction of units, which offer
greater conjugation to the structure, such as indazole rings, these did not improve
their luminescent properties as expected.
All data suggest that Tb(III) ion is better sensitized by both families of ligands than
Eu(III) although ligand Lpzc shows similar quantum yield for both ions and might act
as a promising long-lived luminescent dual probe in multiplexing analysis.
5. Experimental
5.1. General
1H-NMR, 13C-NMR: Bruker AV-300 spectrometer (Departamento de Química
Orgánica, QO). MS: VG Autospec in EI FAB and FAB-HRMS modes (L-SIMS+),
ABSciex QSTAR (ESI+, HRMS), Bruker ULTRAFLEX III (MALDI-TOF/TOF) (HRMS)
spectrometers (Servicio Interdepartamental de Investigación, SIdI). Elemental
analyses : Perkin-Elmer CHN 2400 automatic analyzers (SIdI). All reagents used
were of reagent grade, the solvents were purified prior to their use. Lanthanide
chlorides were purchased from Aldrich and used as received. Absorption spectra:
JASCO V-660 spectrophotometer (QO). Excitation and emission spectra: Varian
Cary Eclypse (SIdI), Jasco FP-8600 (QO), L50 Perkin-Elmer spectrofluorimeters
(QO). The excitation spectra were automatically corrected and the emission spectra
were corrected according to the instruments’ guidebooks. The emission quantum
yields were measured by a relative method using Cs3[Eu(dpa)3] (Φ = 0.24) as a
standard according to literature [25].
15

5.2. General Synthetic Procedures.
Microwave assisted Suzuki-Miyaura cross coupling reaction with boronic acids.
A Microwave vial under argon containing 1a or 1b (0.5mmol), the corresponding
boronic acid (0.51mmol) and Pd(PPh3)4 (0.02mmol) dissolved in anhydrous THF (3.5
mL) was stirred at room temperature for 10 min, then a solution of 0.5mL KOH (1M)
was added to the mixture. The mixture was heated at 130°C for 11 min. in a
Microwave oven (Biotage Initiator 2.5). The solvent was removed under vacuum to
yield a black residue which was dissolved in 20 mL of CH2Cl2 and washed with 10
mL of water. The aqueous phase was extracted with CH2Cl2 (2x20 mL). The
combined organic phases were washed with brine (20 mL), dried over Na2SO4,
filtered and concentrated to dryness. Flash chromatography in CH2Cl2:hexane 1:1
yield the corresponding 2aa-c or 2ba-c compounds.
Microwave assisted Sonogashira cross coupling reaction with Phenylacetylene.
In a Microwave vial under argon containing 1a or 1b (0.46mmol), PdCl2 (PPh3)2
(0.061 eq.) and CuI (0.061 eq.) dissolved in a mixture of anhydrous THF (2.5mL) and
freshly distilled NEt3 (2.2mL), was stirred for 5 minutes. Then phenylacetylene (1.2
eq.) was added and the mixture was heated at 120°C for 13 min. in a Microwave
oven (Biotage Initiator 2.5). The solvent was removed under reduced pressure to
yield a dark residue which was dissolved in 20 mL of CH2Cl2 and washed with 15 mL
of water. The aqueous phase was extracted with CH2Cl2 (2x20 mL). The combined
organic phases were washed with brine (20 mL), dried over Na2SO4, vacuum filtered
and concentrated to dryness. Flash column chromatography, CH2Cl2:hexane 9:1,
yield the corresponding 2ac or 2bc compounds.
Diesters Reduction. In a similar manner as described in [6, 12, 18], LiAIH4, (3 equiv.)
was added in small portions to a soln. of diester (1 equiv.) in 40 ml of THF at 0ºC.
16

The mixture was stirred at r.t. for 90 min and treated with 0.18 ml of H2O, 0.18 ml of
15 % NaOH, and 0.55 ml of H2O. The mixture was filtered and the filtrate
concentrated yielding the diol.
Bromination of bishydroxymethyl derivatives. In a similar manner as described in [6,
12, 18], PBr3, (ca. 5 equiv.) was added to a soln. of diol (1 equiv.) in 60 ml of MeCN
at 40ºC. The mixture was refluxed for 90 min and the solvent removed. The residue
was treated with 10 ml of a sat. soln. of Na2CO3 and extracted with CH2Cl2 (2 x 50
ml). The organic phase was washed with water (3 x 50 ml) and dried over Na2SO4
and concentrated to dryness to yield the expected bisbromomethyl derivatives with
enough purity (NMR) to be used in the next step without further manipulation.
Reaction of Bisbromomethyl compounds with Di(tert-butyl)Iminodiacetate. In a similar
manner as described in [6, 12, 18], a mixture of Na2CO3, (ca. 9 equiv.) and the
bisbromomethyl derivative (1 equiv.) in 40 ml of dry MeCN was added to a soln. of
di(tert-butyl)iminodiacetate (2 equiv.) in 12 ml of dry MeCN. The mixture was stirred
for 24h at r.t. and filtered. The filtrate was evaporated to dryness and dissolved in 25
ml of CH2Cl2. The org. soln. was washed with H2O and worked up as usual. The
resulting oily residue was purified by flash chromatography (AcOEt/hexane 1:3).
Cleavage of Tetra(tert-butyl)esters. In a similar manner as described in [6, 12, 18],
CF3COOH (ca. 9 equiv.) was added to a soln. of tetraester (1 equiv.) in CH2Cl2. The
mixture was stirred for 4 h at r.t. and the solvent removed. The resulting oil was
triturated several times in Et2O and washed once with hot MeOH, leading to a white
precipitate.
4-Bromo-2,6-bis-(3-diethoxycarbonyl-1-pyrazolyl)pyridine, 1a
In a round bottom Schlenk flask under argon 2,6-difluoro-4-bromopyridyne (1 g,
5.15mmol) and 3-ethoxycarbonyl pyrazole (1.44 g, 10.3mmol) were dissolved in 30
17

mL of dry THF. The solution was cooled to 0ºC and NaH (60% in oil, 452 mg,
11.3mmol) was added. The reaction mixture was left at rt. for 4 h, after which 20 mL
of CH2Cl2 was added. The organic phase was washed with water (20 mL) and the
aqueous phase was extracted with CH2Cl2 (3x10 mL). The combined organic phases
were dried over Na2SO4 and concentrated to dryness to afford a solid that was
purified by flash chromatography (CH2Cl2/hexane 1:1) to a white solid 2g, 89%. 1H-
NMR (CDCl3, 300 MHz) δ: 8.55 (2H, d, J= 2.6 Hz), 8.28 (2H, s), 7.01 (2H, d, J= 2.6
Hz), 4.46 (4H, q, J= 7.1 Hz), 1.44 (6H, t, J= 7.2 Hz).ppm; 13C-NMR (CDCl3, 75 MHz)
δ: 161.7; 149.7; 146.9; 137.4; 128.6; 114.6; 110.7; 61.5; 14.3 ppm. Anal. Calc. for
C17H16BrN5O4: C 47.02, H 3.71, N 16.13, found: C 46.82, H 3.57, N 15.92; MS (EI+):
m/z= 433.0 ([M+H]+, 100%), 435.0 ([M+H]++2, 97%)
4-Bromo-2,6-bis-(3-methoxycarbonyl-1-indazolyl)pyridine, 1b
Analogous as described in [16], in a round bottom Schlenk flask under argon 2,6-
difluoro-4-bromopyridyne (680mg, 3.5mmol) and 3-methoxycarbonyl indazol (1.46 g,
8.3mmol) were dissolved in 30mL of freshly distilled DMF. The solution was cooled to
0ºC and NaH (60% in oil, 364mg, 9.1mmol) was added. The reaction mixture was left
at rt then heated at 40ºC for 5 h, after which a white precipitate formed. The solid was
isolated by centrifugation and addition of water to the liquid phase gave a second
precipitate, isolated by centrifugation. The combined solids were washed with of
warm hexane (40mL) and water (40mL), 2.19g, 81%. 1H-NMR (CDCl3, 300 MHz) δ:
8.57 (2H, d, J= 8.6 Hz), 8.31 (2H, d, J=7.8 Hz), 8.24 (2H, s), 7.51 (2H, t, J=8.6 Hz),
7.42 (2H, t, J=7.8 Hz), 4.08 (3H, s) ppm.; 13C-NMR (CDCl3, 75 MHz) δ: 162.1, 151.9,
140.0, 139.2, 136.5, 128.8, 124.8, 125.1, 122.5, 115.8, 114.7, 52.5 ppm. FAB-HRMS
m/z found: 506.0468, calculated for C23H17BrN5O4: 506.0464.
5.3 Luminescence Measurements
18

The luminescence properties of Eu(III), Tb(III) and Gd(III) chelates were measured
using equimolecular mixtures of the ligands Lpza-d and Linda-d, and the
corresponding LnCI3 in borate buffer (pH 8.1). The concentrations used were
between 10-5 and 10-6 mol dm3. In some cases a 2% attenuator in the emission path
was necessary in order to avoid signal saturation, and to keep concentration of
sample and standards in the same range. The energy measurement of the triplet
excited state was determined from the phosphorescence spectra of the Gd(III)
complexes at 77K.
6. Supporting Information
Full synthetic procedures and characterization details for the final products and their
intermediates. Tables of photophysical data of Eu and Tb complexes of Ligands Lpza-
d and Linda-d.
Acknowledgements
The research leading to these results has received funding from the European Union
Seventh Framework Programme (FP7/2007-2013) under Grant Agreement no.
259848. Indirect funding from ERCROS S.A. (Aranjuez, Spain) through the UAM-
ERCROS Chair for Pharmaceutical Chemistry is gratefully acknowledged.
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