Two macrocyclic pentaaza compounds containing pyridine evaluated as novel chelating agents in copper(II) and nickel(II) overload.
ABSTRACT Two pentaaza macrocycles containing pyridine in the backbone, namely 3,6,9,12,18-pentaazabicyclo[12.3.1]octadeca-1(18),14,16-triene (pyN(5)), and 3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1(19),15,17-triene (pyN(5)), were synthesized in good yields. The acid-base behaviour of these compounds was studied by potentiometry at 298.2K in aqueous solution and ionic strength 0.10 M in KNO(3). The protonation sequence of pyN(5) was investigated by (1)H NMR titration that also allowed the determination of protonation constants in D(2)O. Binding studies of the two ligands with Ca(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Pb(2+) metal ions were performed under the same experimental conditions. The results showed that all the complexes formed with the 15-membered ligand, particularly those of Cu(2+) and especially Ni(2+), are thermodynamically more stable than with the larger macrocycle. Cyclic voltammetric data showed that the copper(II) complexes of the two macrocycles exhibited analogous behaviour, with a single quasi-reversible one-electron transfer reduction process assigned to the Cu(II)/Cu(I) couple. The UV-visible-near IR spectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonal distorted coordination geometry for the metal centre. X-band EPR spectra of the copper(II) complexes are consistent with distorted square pyramidal geometries. The crystal structure of [Cu(pyN(5))](2+) determined by X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in a distorted square pyramidal environment.
Two macrocyclic pentaaza compounds containing pyridine evaluated as novel
chelating agents in copper(II) and nickel(II) overload
Ana S. Fernandesa, M. Fátima Cabrala, Judite Costaa,⁎, Matilde Castroa, Rita Delgadob,c,
Michael G.B. Drewd, Vitor Félixe
aiMed.UL, Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
bInstituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
cInstituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
dSchool of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD UK
eDepartamento de Química, CICECO, and Secção Autónoma de Ciências da Saúde, Universidade de Aveiro, 3810-193 Aveiro, Portugal
a b s t r a c t a r t i c l ei n f o
Received 1 July 2010
Received in revised form 17 November 2010
Accepted 19 November 2010
Available online xxxx
Two pentaaza macrocycles containing pyridine in the backbone, namely 3,6,9,12,18-pentaazabicyclo[12.3.1]
octadeca-1(18),14,16-triene (pyN5), and 3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1(19),15,17-
triene (pyN5), were synthesized in good yields. The acid–base behaviour of these compounds was
studied by potentiometry at 298.2 K in aqueous solution and ionic strength 0.10 M in KNO3. The protonation
sequence of pyN5 was investigated by
protonation constants in D2O. Binding studies of the two ligands with Ca2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+
metal ions were performed under the same experimental conditions. The results showed that all the
complexes formed with the 15-membered ligand, particularly those of Cu2+and especially Ni2+, are
thermodynamically more stable than with the larger macrocycle. Cyclic voltammetric data showed that the
copper(II) complexes of the two macrocycles exhibited analogous behaviour, with a single quasi-reversible
one-electron transfer reduction process assigned to the Cu(II)/Cu(I) couple. The UV–visible-near IR
spectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonal
distorted coordination geometry for the metal centre. X-band EPR spectra of the copper(II) complexes are
consistent with distorted square pyramidal geometries. The crystal structure of [Cu(pyN5)]2+determined
by X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in a
distorted square pyramidal environment.
1H NMR titration that also allowed the determination of
© 2010 Elsevier Inc. All rights reserved.
The therapy for metal overload pathologies usually involves the
administration of suitable chelators to selectively remove the metal from
the body. Regarding copper(II) and nickel(II) metal ions, there is still a
need for safe and efficient chelating agents, as the existing ones have
a number of drawbacks such as toxic side effects and controversial
Copper as an essential element is a component of many metallopro-
teins and enzymes and plays a vital role in electron transfer reactions
of many cellular processes. However, excessive copper can be very
toxic resulting in severe diseases . Certain chelating agents have been
shown to bind copper with high affinity. Previous work on copper(II)
chelation agents has focused on Wilson's disease, which is an inherited
metabolic disease of copper toxicity that is fatal if left untreated .
D-Penicillamine has been one of the most commonly used chelating
agents for treatment of this disease. When the patient cannot tolerate
treatment with D-penicillamine, trien [N,N′-bis(2-aminoethyl)ethane-
1,2-diamine] and ammonium tetrathiomolybdate are considered
safer alternatives. Trien is a lesser active agent for copper(II) removal
in biological media than D-penicillamine, and although both chelators
have similar toxicity, side effects are less frequent and generally
milder with D-penicillamine. Ammonium tetrathiomolybdate, acting
differently from both D-penicillamine and trien, has been used due to
its lower toxic profile, but it is still an experimental drug and its long-
term efficacy is unknown .
Copper(II) chelation therapy attracts also attention in recent
investigations and treatment of neurodegenerative disorders, such as
Alzheimer, Parkinson, and Creutzfeldt–Jakob . Furthermore, an
excess of copper appears to be an essential co-factor for angiogenesis.
Moreover, high levels of copper were found in many human cancers,
including prostate, breast, colon, lung, and brain. Consequently, the
therapeutic value of copper(II) chelators as anti-angiogenic molecules
in the treatmentof these cancers hasbeen reported. More recently,
mixtures of copper(II) chelators and copper salts were found to act as
efficient proteasome inhibitors and apoptosis inducers, specifically in
cancer cells .
Journal of Inorganic Biochemistry 105 (2011) 292–301
⁎ Corresponding author. Fax: +351 217 946 470.
E-mail address: email@example.com (J. Costa).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
On the other hand, human exposure to nickel occurs primarily via
to adverse effects on human health. Nickel allergy in the form of contact
dermatitis is the most common and well-known reaction. Although the
accumulation of nickel in the body through chronic exposure can cause
lung fibrosis, kidney, and cardiovascular diseases, the most serious
concerns relate to nickel's carcinogenic activity. Epidemiological studies
have clearly implicated nickel compounds as human carcinogens [8,9].
All nickel compounds, except for metallic nickel, were classified as
carcinogenic tohumans in 1990 by the International Agency for Research
on Cancer (IARC) .
Over the years, various chelators have been investigated for their
ability to bind nickel. The most effective ones are EDTA, DTPA
(diethylenetriaminepentaacetic acid), diethyldithiocarbamate, tet-
raethylthiuram disulfide, and clioquinol (5-chloro-8-hydroxy-7-
iodoquinoline), all of them presenting considerable side effects
Therefore, thedevelopmentof novel chelators selective for nickel(II)
and for copper(II), and exhibiting minor side effects is an imperative
research. This led us to investigate the possible use of macrocyclic
that are particularly different from those of analogous open chain
chelators. Macrocycles having more rigid structures can impose specific
coordination geometry to the metal ion, whereas open chain chelators
adapt more easily to the geometric requirements of the metal centre
. In the present work, the synthesis and characterization of two
pentaaza macrocyclic compounds containing pyridine in the backbone,
ene) and pyN5 (3,6,10,13,19-pentaazabicyclo[13.3.1]nonadeca-1
(19),15,17-triene), cf. Scheme 1, as well as the study of their copper(II)
and nickel(II) complexes are reported, in order to evaluate their possible
these two macrocycles was studied and their ability to coordinate Cu2+
and Ni2+and other divalent metal ions (Ca2+and Zn2+are included due
to their essential role in living organisms) was evaluated. The adopted
structures of the Cu(II) and Ni(II) complexes were also studied by
spectroscopic methods insolution,andthesingle crystalX-raydiffraction
of [CupyN5](PF6)2was determined. Finally, due to the important role
of the redox behaviour of the copper(II) complexes in biology some
voltammetric studies were carried out.
2. Experimental section
2.1. General procedures
Elemental analysis was performed on a VarioEL CHNS analyser
fromvacuum-driedpowdersamples.Meltingpoints were determined
with a Köpffer Melting Point apparatus.
and N,N′-bis(2-aminoethyl)1,3-propanediamine were purchased from
Aldrich. 2,6-Pyridinedicarbaldehyde was prepared by published methods
. All the commercially available chemicals were of reagent grade and
used as supplied without further purification. Organic solvents were
purified or dried by standard methods .
in the presence of organic matter are potentially explosive and should be
prepared in small quantities.
2.2. Synthesis of the macrocycles
2.2.1. Synthesis of the macrocycle pyN5
To a stirred solution of freshly prepared 2,6-pyridinedicarbalde-
hyde (2.34 g, 18 mmol) in methanol (40 mL) was added a solution of
Pb(NO3)2(6.1 g, 18 mmol) in water (80 mL). To the resulting solution
was added dropwise, with rapid stirring, a solution of N,N′-bis(2-
aminoethyl)ethane-1,2-diamine (3.35 g, 18 mmol) in methanol
(40 mL) over a period of 3 h. The solution was stirred while heating
under reflux for 7 h, during which time an intense deep red colour
developed. After reflux the solution was cooled to 5 °C, and sodium
borohydride (4.36 g, 45.5 mmol) was added in small portions over
60 min.The yellowsolutionobtainedwasstirredat roomtemperature
for 30 min and then heated on a hot water-bath at 60 °C for 30 min,
treating the mixture with Na2S.9H2O (10 g, 42 mmol) followed by
heating on a hot water-bath for 30 min. The solution was then cooled,
The filtrate was extracted with dichloromethane (4×50 mL), the
combined extracts were dried with anhydrous MgSO4, and the dichlor-
omethane was removed with a rotary evaporator to leave a light yellow
oil. This oil was dissolved in methanol and 37% hydrochloric acid was
added until pH≈2. During the addition, an off-white solid precipitated,
whichwas identifiedas thepuredesired compound. Yield: 85%.Mp280–
282 °C (decomp.).1H NMR (D2O, pD=5.10): δ 3.21 (4H, s (singlet), N–
CH2), 3.41 (4H, t (triplet), N–CH2–CH2–N), 3.51 (4H, t, N–CH2–CH2–N),
4.57 (4H, s, N–CH2–py), 7.53 (2H, d (doublet), py) and 7.99 (1H, t, py)
ppm.13C NMR (D2O, pD = 5.10): δ 43.92 (N–CH2–CH2–N), 45.64 (N–
and 150.94 (py) ppm. Found: C, 37.03; H, 7.16; N, 16.56. Calc. for
C13H23N5·4HCl·1.5H2O: C, 36.98, H, 7.16, N, 16.59%.
2.2.2. Synthesis of the macrocycle pyN5
A procedure analogous to that described for pyN5was used,
replacing N,N′-bis(2-aminoethyl)ethane-1,2-diamine by N,N′-bis(2-
aminoethyl)1,3-propanediamine. The product was obtained as a thick
yellow oil, which was purified by passing through a neutral alumina
column (2.5×30 cm) and eluting with dichloromethane–methanol
(10:0.5 v/v). The pure compound was dissolved in methanol and 37%
Yield: 46%. Mp 266–268 °C (decomp.).1H NMR (D2O, pD=2.55): δ 2.21
(2 H, q (quintuplet), CH2–CH2–N), 3.37 (4H, t, CH2–CH2–N), 3.65 (4H, t,
N–CH2–CH2–N), 3.71 (4H, t, N–CH2–CH2–N), 4.63 (4H, s, N–CH2–py),
7.56 (2H, d, py) and 8.00 (1H, t, py) ppm.13C NMR (D2O, pD = 2.55): δ
21.30 (CH2–CH2–N), 42.25 (N–CH2–CH2–N), 43.20 (N–CH2–CH2–N),
44.14 (CH2–CH2–N), 51.35 (N–CH2–py), 124.50 (py), 140.44 (py) and
150.68 (py) ppm. Found: C, 37.59; H, 7.80; N, 15.37. Calc. for C14H25-
N5·4HCl·2H2O: C, 37.80, H, 7.50, N, 15.70%.
A.S. Fernandes et al. / Journal of Inorganic Biochemistry 105 (2011) 292–301
2.2.3. Synthesis of the copper(II) complex [CupyN5](PF6)2
An aqueous solution of Cu(ClO4)2.6H2O (0.150 mmol, 0.056 g) was
added to a stirred solution of pyN5(0.150 mmol, 0.0593 g) dissolved
intheminimumvolumeofwater(≈1 mL).Then0.0489 g(0.300 mmol)
of NH4PF6wasadded and themixturewasstirredat 60 °C for 1 h.The pH
minimum amount of methanol–acetonitrile (10:2.5). Blue crystals were
formed in about 4 weeks by slow evaporation of the solvent mixture at
4 °C. Yield: ≈ 80%.
2.3. Potentiometric measurements
2.3.1. Reagents and solutions
Stock solutions of the ligands were prepared at ca. 2.50×10−3M.
Metal ion solutions were prepared atabout 0.025to0.050 M from nitrate
salts (analytical grade) in demineralized water (from a Millipore/Milli-Q
system) and were standardized by titration with Na2H2EDTA .
Carbonate-free solutions of the titrant, KOH, were prepared at ca.
0.10 M by dilution of a commercial ampoule of Titrisol (Merck) with
demineralized water under a stream of pure argon gas. These solutions
HNO3prepared from a Merck ampoule was used. The titrant solutions
were standardized (tested by Gran method) . For the competition
titrations, a standard K2H2EDTA aqueous solution was used.
2.3.2. Equipment and work conditions
The potentiometric setup for conventional titrations consisted of a
50 mL glass-jacketed titration cell sealed from the atmosphere and
connected to a separate glass-jacketed reference electrode cell by a
Wilhelm-type salt bridge containing 0.10 M KNO3solution. An Orion
720A+measuring instrument fitted with a Metrohm 6.0150.100 glass
electrode and a Metrohm 6.0733.100 Ag–AgCl reference electrode was
used for the measurements. The ionic strength was kept at 0.10±0.01 M
with KNO3, temperature was controlled at 298.2±0.1 K by circulating
water through the jacketed titration cell using a Huber Polystat cc1
thermostat, and atmospheric CO2was excluded from the titration cell
during experiments by passing argon across the top of experimental
of the experimental solution by a Metrohm Dosimat 765 automatic
burette. Titration procedure was automatically controlled by software
after selection of suitable parameters, allowing for long unattended
The [H+] of the solutions was determined by the measurement of
the electromotive force of the cell, E=E'o+Q log[H+]+Ej. The term
pH is defined as −log [H+]. E'oand Q were obtained by titrating a
solution of known hydrogen-ion concentration at the same ionic
strength, using the acid pH range of the titration. The liquid-junction
potential, Ej, was found to be negligible under our experimental
conditions. The value of Kwwas determined from data obtained in the
alkaline range of the titration, considering E'oand Q validfor the entire
pH range and found to be equal to 10−13.80M2. The potentiometric
equilibrium measurements were carried out using 20.00 mL of ca.
2.50×10−3M ligand solutions diluted to a final volume of 30.00 mL,
in the absence of metal ions and in the presence of each metal ion for
which the CM:CLratio was 1:1. For the reactions of Cu2+with both
ligands, competition titrations were performed. K2H4EDTA was used
as the reference ligand, for which values of protonation and stability
constants were determined before under the same experimental
conditions: log K1
log KCuEDTA=19.23, log KCuHEDTA=3.06, log KCuEDTAOH=11.33 .
Ratios of 0.75:1:1 and 1:1:1 (CL:CL':CCu) were used for L=pyN5
and pyN5, respectively, and L′=EDTA. The competition reactions
H=10.22, log K2
H=6.16, log K3
H=2.71, log K4
reached equilibrium upon 15 to 20 min at each point in the pH range
where the competition reaction took place. The same values for the
stability constants were obtained in both directions of the reaction,
the direct curve titrating with KOH and the back titration with HNO3.
2.3.4. Calculation of equilibrium constants
Overall equilibrium constants βi
[MmHhLl]/[M]m[H]h[L]l) were calculated by fitting the potentiometric
data from protonation or complexation titrations with the HYPERQUAD
program . Species distribution diagrams were plotted from the
calculated constants with the HYSS program . Only mononuclear
species, ML, MHL, and MH-1L were found for the metal complexes of the
values of βMHL(or βMH-1 L) and βMLconstants provide the stepwise
reaction constants. The species considered in a particular model were
those that could be justified by the principles of coordination chemistry.
The errors quoted are the standard deviations of the overall stability
constants given directly by the program for the input data, which include
all the experimental points of all titration curves, and determined by the
normal propagation rules for the stepwise constants.
Protonation constants were obtained from ca. 180 experimental
points, and stability constants for each metal ion were determined
from 120 to 180 experimental points (2 or 3 titration curves).
Hand βMmHhLl (being βMmHhLl=
2.4. NMR measurements
2.4.1. Characterization of the macrocycles
recorded on a Bruker Avance-400 spectrometer at 294 K probe
temperature. Chemical shifts (δ) were given in ppm and coupling
constants (J) in Hz. The NMR spectra were performed in CDCl3(δ ppm
1H: 7.26;13C: 77.16) or in D2O. The reference used for the1H NMR
measurements in D2O was 3-(trimethylsilyl)propionic acid-d4-sodium
salt (DSS) and in CDCl3the solvent itself (at 7.26 ppm). For13C NMR
spectra 1,4-dioxane (δ ppm:1H: 3.75;13C: 67.20) was used as internal
reference. 2D NMR spectra correlation spectroscopy (COSY), hetero-
nuclear multiple quantum coherence (HMQC), and heteronuclear
multiple bond correlation (HMBC) were acquired using gradient pulse
programs from Bruker library. Phase-sensitive nuclear Overhauser effect
spectroscopy (NOESY) was performed using a mixing time of 1.5 s. Two
and monodimensional FIDs were processed using the TopSpin software
and multiplicity for1H spectra and on 2D experiments for13C spectra.
1H (400.13 MHz) and
13C NMR (100.62 MHz) spectra were
2.4.2. NMR titration measurements
The titration of pyN5(0.010 M in D2O) was carried out in the
NMR tube. The pD values were adjusted by adding DCl or CO2-free
KOD solutions. The −log [H⁎] was measured directly in the NMR tube
with a combined glass Ag–AgCl microelectrode (Mettler-Toledo
U402-M3-S7/200) coupled with an Orion 3 Star pH meter. The
electrode was previously standardized with commercial aqueous
buffer solutions, and the pD values were calculated according to the
equation pD=pH⁎+(0.40±0.02), where pH* is the direct pH
reading . The dissociation constants in D2O (pKD) were calculated
from the NMR titration data, using a non-linear least-squares curve-
fitting procedure that minimizes the sum of the squares of the
deviations of the observed and calculated values of the chemical
shifts. These pKDvalues were converted to pKHvalues obtained in
water by the equation pKD=0.11+1.10×pKH.
2.4.3. Magnetic moments
Magnetic moments were measured at 294 K using solutions of Ni
6.32) in D2O. The1H NMRspectra ofthe solutionswithDSS,asinternal
reference, were acquired in a tube containing an internal capillary
2+(2.38×10−2M, pH 6.45) and NipyN5
A.S. Fernandes et al. / Journal of Inorganic Biochemistry 105 (2011) 292–301
filled with D2O and DSS, and the corresponding magnetic moments
calculated from the shift (Δδ) between both reference signals .
2.5. Spectroscopic studies
Electronic spectra were recorded with a UNICAM model UV-4
(UV–visible) or a Shimadzu model UV-3100 (UV–visible-near IR)
spectrophotometers using aqueous solutions of Ni2+and Cu2+
complexes of both macrocycles (1.0×10−2to 1.0×10−3M) at pHs
6.63 to 7.05.
EPR spectroscopy measurements of copper(II) complexes of 
pyN5and pyN5were recorded at 99 K with a Bruker EMX 300
spectrometer equipped with continuous-flow cryostats for liquid
nitrogen, operating at X-band. The complexes were prepared at about
5.04, 7.23, and 9.92 for CupyN5
2+, in 1 M NaClO4aqueous solution.
2.6. Electrochemical studies
A BAS CV-50W Voltammetric Analyzer connected to BAS/Win-
dows data acquisition software was used. Cyclic voltammetric
experiments were performed in a glass cell MF-1082 from BAS in a
C-2 cell enclosed in a Faraday cage, at room temperature, under argon.
The reference electrode was Ag–AgCl (MF-2052 from BAS) filled with
NaCl 3 M in water, standardized for the redox couple Fe(CN)6
1032 from BAS) with a gold-plated connector. The working electrode
was a glassy carbon (MF-2012 from BAS).
Copper(II) complexes of pyN5and pyN5(1.63×10−3M;
pH=7.05 and 1.46×10−3M; pH=7.09, respectively) were prepared
in 0.1 M KNO3in water. The solutions were deaerated by an argon
stream prior to all measurements and were kept under argon during
the measurements. Between each scan, the working electrode was
electrocleaned by multi-cycle scanning in the supporting electrolyte
solution, polished on diamond 1 μm and on alumina 0.3 μm, cleaned
with water and sonicated before use, according to standard
Cyclic voltammograms with sweep rate ranging from 25 to
1000 mV s−1were recorded in the region from +1.2 to −1.2 V. At
this potential range the ligands were found to be redox inactive. The
half-wave potentials, E1/2, were obtained by averaging the anodic and
cathodic peak potentials. All potential values are reported relative to
the Ag–AgCl reference electrode and the E1/2and ΔEpof the Fe(CN)6
and 73 mV, respectively.
4−. The auxiliary electrode was a 7.5-cm platinum wire (MW-
4−couple, under our experimental conditions, were 196 mV
2.7. X-ray crystallography
Blue crystals of [CupyN5](PF6)2with suitable quality for single
crystal X-ray diffraction determination were grown up from metha-
Crystal data: C13H21CuF12N5, Mr=600.83; monoclinic, space
group P21/c, Z=4, a=8.8619(9) b =14.9388(14), c=16.6689(16)
Å, β =103.674(9)°, U=2144.2(4) Å3, ρ(calc)=1.861 Mg m−3, μ(Mo-
Kα) = 1.283 mm−1.
using graphite monocromatized Mo-Kα radiation (λ=0.71073 Å) at
Reading University. The selected crystal was positioned at 50 mm from
the CCD, and the frames were taken using a counting time of 2 s. The
processing of the data was carried out with the Crysalis program .
Intensities were corrected for empirical absorption effects with the
ABSPACK program . The structure was solved by direct methods and
squares on F2using the SHELX-97 suite . Anisotropic thermal
parameters were used for the non-hydrogen atoms. The hydrogen
in calculated positions with isotropic parameters equivalent to 1.2 times
those of the atom to which they were attached. The final refinement of
298 parameters converged to final R and Rwindices R1=0.0467 and
wR2=0.1028 for 2861 reflections with IN2σ(I) and R1=0.0992 and
wR2=0.1078 for all 6262 hkl data. Molecular diagrams presented are
drawn with graphical package software PLATON .
3. Results and discussion
3.1. Synthesis and characterization of the macrocycles
Compounds pyN5and pyN5were prepared in good yield
by [1:1] condensation of 2,6-pyridinedicarboxaldehyde and N,N′-bis
(2-aminoethyl)ethane-1,2-diamine (trien) and N,N′-bis(2-ami-
noethyl)1,3-propanediamine, respectively, using Pb2+as the tem-
plate ion, followed by reduction of the resulting tetraimines with
sodium borohydride. The pure products were obtained as tetrahy-
drochloride salts in 85% and 46% yields, respectively. The lower yield
of the later compound results from the unfavourable adopted
geometry of the lead(II) complex during the cyclization reaction .
However, Ca2+or Ba2+did not lead to better yields.
Both macrocycles were synthesized by different and more time
consuming procedures [28–30]. Stetter et al.  prepared pyN5
followinga modified Richmanand Atkinsmethod  in78% yield, and
Riley et al.  followed the same procedure with minor changes.
Kimura et al.  prepared pyN5, in unspecified yield, by refluxing
the bisdiethyl esters of pyridine-2,6-dicarboxylic acid and N,N′-bis(2-
aminoethyl)1,3-propanediamine in ethanol and high dilution followed
by reduction of the resulted diamide with diborane in tetrahydrofuran.
1D and 2D NMR spectroscopy were used for characterization of 
pyN5and pyN5. The chemical shifts and the corresponding assign-
ments were accomplished by1H,13C, COSY, HMQC, HMBC, and NOESY at
pD 5.10 and 2.55, respectively, as described in Appendix A of the
Supplementary material (cf. Table S1 and Figs. S1–S5).
3.2. Acid–base behaviour of the ligands
The acid–base behaviour of pyN5and pyN5was studied by
potentiometry in water at 298.2 K and ionic strength 0.10 M in KNO3.
The former compound was also studied by1H NMR spectroscopy. The
determined protonation constants are collected in Table 1 together
with the values of the related aneN5and aneN5compounds
(cf. Scheme 1) for comparison. Both compounds have five basic
centres; however, only three constants for pyN5and four for 
pyN5could be accurately determined by potentiometry and one more
for pyN5was obtained by1H NMR. The two compounds exhibit
high and fairly high values, respectively, for the first two protonation
constants corresponding to the protonation of nitrogen atoms in
opposite positions, minimizing the electrostatic repulsion between
positive charges of the ammonium groups formed. The third and
fourth constants are much lower due to the stronger electrostatic
repulsions as they correspond to protonation of nitrogen atoms at
short distances from already protonated ones and to the limited
motion allowed in the ring backbone. The increase in basicity of these
two last centres in pyN5is correlated with the increase of the
length of the chain between contiguous nitrogen atoms. The values
reported before (in NaClO4medium) [29–33] shown in Table 1 differ
slightly from ours; however, for the first time, we were able to
accurately determine the fourth protonation constant.
The overall basicity and all the stepwise protonation constants of 
corresponding macrocycles without pyridine as expected taking into
account the electron withdrawing effect of the pyridine ring.
1H NMR spectroscopic titration of pyN5was carried out in
order to understand its protonation sequence and to determine the
lower protonation constants. In Fig. 1 is shown the spectrum of the
A.S. Fernandes et al. / Journal of Inorganic Biochemistry 105 (2011) 292–301
ligand at pD 5.10 and the titration curves for all resonances. The1H
NMR spectrum exhibits six resonances in the 1.24–7.04 pD region, but
for higher pD values, Hdand Heresonances overlap. The resonances at
7.99 and 7.53 ppm were assigned to Haand Hdprotons, the two
singlets at 4.57 and 3.21 ppm to Hcand Hfprotons and the triplets at
3.51 and 3.41 ppm to Hdand Heprotons, respectively.
Stepwise protonation constants (log Ki
H) of pyN5, pyN5and other similar compounds for comparison.aT=298.2 K; I=0.10 M in KNO3.
Equilibrium quotient pyN5
aValues in parentheses are standard deviations on the last significant figure.
bT=298.2 K; I=0.2 M in NaClO4; ref. .
cT=298.2 K; I=0.1 M in NaClO4; ref. .
dT=298.2 K; I=0.1 M NaClO4; ref. .
eT=298.2 K; I=0.2 M in NaClO4; ref. .
fDetermined in this work by1H NMR spectroscopy, using the calculated value of pKD4and the equation pKD=0.11+1.10×pKH.
Fig. 1. (a)1H NMR titration curves for pyN5, chemical shift δH(ppm) in function of pD; (b)1H NMR spectrum of pyN5(D2O, pD 5.10).
A.S. Fernandes et al. / Journal of Inorganic Biochemistry 105 (2011) 292–301