Chemical, radiochemical and biological studies of new gallium(iii) complexes with hexadentate chelators

Article abstract of DOI:10.1039/c4dt02274b

New N₄O₂-donor acyclic chelators 2-[[2-[2-(3,5-dimethylpyrazol-1-yl)ethyl-[2-[(2-hydroxyphenyl)methylamino]ethyl]amino]ethylamino]methyl]phenol (H₂L^pz,NH) and 2-[[2-[2-[(2-hydroxyphenyl)methylamino]ethyl-(2-pyri

Full text of DOI:10.1039/c4dt02274b

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Chemical, Radiochemical and Biological Studies of New Gallium(III)
Complexes with Hexadentate Chelators
Francisco Silva1, Maria Paula C. Campello1, Lurdes Gano1, Célia Fernandes1, Isabel C.
Santos1, Isabel Santos1, José R. Ascenso2, M. João Ferreira2, António Paulo1,*
1Centro de Ciências e Tecnologias e Nucleares, IST, Universidade de Lisboa, Estrada
Nacional 10, 2695ꢀ066 Bobadela LRS, Portugal
2Centro de Quimica Estrutural, IST, Universidade de Lisboa, Avenida Rovisco Pais 1,
1049ꢀ001, Lisboa, Portugal
Keywords: Gallium, Acyclic Chelators, Xꢀray Structures, NMR Studies,
Radiopharmaceuticals
* eꢀmail: apaulo@ctn.ist.utl.pt
Dalton Transactions: submitted as a full paper
1

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Abstract
New N4O2ꢀdonor acyclic chelators 2ꢀ[[2ꢀ[2ꢀ(3,5ꢀdimethylpyrazolꢀ1ꢀyl)ethylꢀ[2ꢀ[(2ꢀ
hydroxyphenyl)methylamino]ethyl]amino]ethylamino]methyl]phenol (H2Lpz*,NH) and
2ꢀ[[2ꢀ[2ꢀ[(2ꢀhydroxyphenyl)methylamino]ethylꢀ(2ꢀ
pyridylmethyl)amino]ethylamino]methyl]phenol (H2Lpy,NH
)
were obtained upon
introduction of pyridyl or pyrazolyl coordinating units at the central nitrogen atom of
diethylenetriamine (dien) and by functionalization of its terminal amines with phenol
groups. The coordination behavior of H2Lpz*,NH and H2Lpy,NH was evaluated towards
natGa/67Ga, aiming to assess their suitability to obtain Ga(III) chelates relevant for
biomedical applications. Single crystal Xꢀray diffraction analysis of the complexes
[GaLpz*,NH](ClO4) and [GaLpy,NH](ClO4), confirmed the presence of N4O2ꢀhexadentate
chelators with the phenoxide groups coordinated cis relatively to the pyridyl/pyrazolyl
arms. Unlike [GaLpz*,NH](ClO4), [GaLpy,NH](ClO4) exists in solution as a mixture of
isomers, as confirmed by several NMR techniques. The corresponding radiocomplex
[67GaLpy,NH +
was obtained smoothly in almost quantitative radiochemical yield,
]
contrarily to [67GaLpz*,NH]+ that seems to be (hemi)labile under the same conditions.
[67GaLpy,NH]+ presents a high in vivo stability and a favourable biodistribution profile in
mice. The imine congeners 2ꢀ[(E)ꢀ2ꢀ[2ꢀ(3,5ꢀdimethylpyrazolꢀ1ꢀyl)ethylꢀ[2ꢀ[(E)ꢀ(2ꢀ
hydroxyphenyl)methyleneamino]ethyl]amino]ethyliminomethyl]phenol
and 2ꢀ[(E)ꢀ2ꢀ[2ꢀ[(E)ꢀ(2ꢀhydroxyphenyl)methyleneamino]ethylꢀ(2ꢀ
(H2Lpz*,C=N
)
pyridylmethyl)amino]ethyliminomethyl]phenol (H2Lpy,C=N) were also evaluated but
they did not form complexes compatible with biomedical applications owing to their
high reactivity.
2

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Introduction
Metalꢀbased compounds have an increasing importance in most prominent biomedical
applications, as they offer the possibility of exploring a higher variety of structural
motifs and reactivity patterns if compared with purely organic molecules. Together with
the design of cytotoxic antiꢀcancer drugs, the development of radiopharmaceuticals can
be considered the most important application of coordination and organometallic
complexes of transition and postꢀtransition metals. Radiopharmaceuticals are drugs
containing a radionuclide in their composition and are used in nuclear medicine for
diagnosis and therapy. For diagnosis, there are two modalities, single photon emission
computed tomography (SPECT) and positron emission tomography (PET), which make
use of gammaꢀ or positronꢀemitting radionuclides, respectively.
Gallium complexes have emerged as one of the most promising new classes of
cytotoxic drugs and diagnostic radiopharmaceuticals.1ꢀ6 In particular, galliumꢀ67 and
galliumꢀ68 complexes are suitable for SPECT and PET imaging, respectively.
Moreover, 68Ga is available from the 68Ge/68Ga generator, which allows the possibility
of obtaining a PET radionuclide without needing the presence of a nearby cyclotron.7
This possibility anticipates a bright future for 68Ga in PET imaging, in the same way as
99mTc became the workhorse of SPECT imaging several years ago.
Like for other radiometals, the biodistribution of 67Ga/68Ga complexes to be
evaluated as radiopharmaceuticals depends on their chemical and physical properties –
perfusion radiopharmaceuticals ꢀ or on their biological interactions ꢀ targetꢀspecific
radiopharmaceuticals. The biological distribution of perfusion agents is determined by
blood flow and these agents target high capacity systems, such as phagocytosis,
hepatocyte clearance and glomerular filtration. The targetꢀspecific radiopharmaceuticals
target low capacity systems, and their biodistribution is determined by specific protein
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interactions, for example antigen, enzymatic or receptorꢀbinding interactions.8 Inorganic
chemists play an important role in the design of perfusion or targetꢀspecific 67/68Ga
radiopharmaceuticals, as it is necessary to design and have available biocompatible
chelators that form thermodynamically stable and kinetically inert Ga complexes.
Ga(III) is a rather hard Lewis acid and, for this reason, macrocyclic or acyclic
polyaminocarboxylic ligands (Chart 1) are among the most explored chelators to obtain
stable and inert Ga complexes suitable for the design of radiopharmaceuticals.5,6
Chart 1. Selected examples of acyclic and cyclic chelators used in the design of
perfusion or targetꢀspecific gallium radiopharmaceuticals.
Macrocyclic ligands, like 1,4,7ꢀtriazacyclonanonaneꢀ1,4,7ꢀtriacetic acid (NOTA)
and 1,4,7,10ꢀtetraazacyclonanonaneꢀ1,4,7,10ꢀtetraacetic acid (DOTA), can afford Ga
complexes of high kinetic inertness and thermodynamic stability that show a high
resistance to in vivo transchelation processes.6 These favorable features justify that
bifunctional versions of NOTA and DOTA have been thoroughly applied in the 67Gaꢀ or
4

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68Gaꢀlabeling of clinically relevant biomolecules, namely in the case of biologically
active peptides.3,5 Very good results were obtained for 68GaꢀDOTATOC and 68Gaꢀ
DOTANOC, which are radiometallated analogues of somatostatin (SST) clinically used
to image SSTꢀreceptor positive malignancies.9 However, the synthesis of suitable
macrocyclic bifunctional chelators for the conjugation of the biomolecules and still
capable of stabilizing the metal ion can be considerably challenging, as only one of the
pendant arms must be selectively functionalized with the targeting molecule. For this
reason, several research groups have pursued in recent years with the synthesis and
evaluation of acyclic chelators suitable for the stable complexation of 67Ga and 68Ga
under physiological conditions. Recently, encouraging results have been reported for
linear and potentially hexadentate ligands (e.g. HBED, H2dedpa and CP256 (Chart 1)),
which have been successfully applied in 67Ga/68Gaꢀlabelling of peptides and proteins.10ꢀ
13
Moreover, acyclic chelators might also be useful for the design of galliumꢀbased
perfusion radiopharmaceuticals.14ꢀ17 For instance, several N4O2ꢀdonor Schiff bases of
the bis(salicyladimine) type, namely BAPDMEN derivatives (Chart 1), have been
applied to obtain cationic and lipophilic 67Ga/68Ga complexes for cardiac perfusion
imaging or for in vivo detection of multidrug resistance.15ꢀ17
In the past few years, our group has been involved in the study of Ga(III)
complexes of biomedical relevance, either as cytotoxic drugs or as
radiopharmaceuticals.18,19 Within our interest on galliumꢀbased (radio)pharmaceuticals,
we have embarked on the synthesis of new N4O2ꢀdonor acyclic ligands and we have
evaluated their coordination capability towards the Ga(III) metal ion, aiming to have an
insight on their potential usefulness as hexadentate chelators for radiopharmaceutical
research. These N4O2ꢀdonor ligands (2ꢀ[[2ꢀ[2ꢀ(3,5ꢀdimethylpyrazolꢀ1ꢀyl)ethylꢀ[2ꢀ[(2ꢀ
hydroxyphenyl)methylamino]ethyl]amino]ethylamino]methyl]phenol (H2Lpz*,NH) and
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2ꢀ[[2ꢀ[2ꢀ[(2ꢀhydroxyphenyl)methylamino]ethylꢀ(2ꢀ
pyridylmethyl)amino]ethylamino]methyl]phenol (H2Lpy,NH)), featuring a Nꢀheterocyclic
pendant arm of the pyridyl and pyrazolyl type and two phenolate coordinating groups,
have been designed based on the diethylenetriamine (dien) framework and were
obtained by reduction of the corresponding Schiff bases 2ꢀ[(E)ꢀ2ꢀ[2ꢀ(3,5ꢀ
dimethylpyrazolꢀ1ꢀyl)ethylꢀ[2ꢀ[(E)ꢀ(2ꢀ
hydroxyphenyl)methyleneamino]ethyl]amino]ethyliminomethyl]phenol
(H2Lpz*,C=N
)
and 2ꢀ[(E)ꢀ2ꢀ[2ꢀ[(E)ꢀ(2ꢀhydroxyphenyl)methyleneamino]ethylꢀ(2ꢀ
pyridylmethyl)amino]ethyliminomethyl]phenol (H2Lpy,C=N), respectively. The selected
Nꢀheterocyclic groups have a well documented capacity of coordinating different metal
centers under the aqueous conditions required in radiopharmaceutical chemistry. 11,12,20ꢀ
22
By proposing these ligands, we also took into consideration that they can be obtained
in a modular way in a few steps, by combination of the desired Nꢀheterocyclic and
phenol derivatives. Therefore, these N4O2ꢀdonors are easily tunable upon introduction
of different substituents (e.g. alkyl or ether groups) in the dien backbone and/or in the
different aromatic rings, while offering the possibility of a selective functionalization of
the Nꢀheterocyclic rings with groups (e.g. –COOH) suitable for further coupling of
bioactive molecules. We have anticipated that this versatile tunability would help on the
modulation/optimization of the physicoꢀchemical properties of the respective Ga(III)
complexes as perfusion radiopharmaceuticals, e.g for myocardial imaging, as well as on
their use as potential bifunctional chelators for the design of targetꢀspecific
radiopharmaceuticals.
In this manuscript, it is reported the synthesis of H2Lpz*,NH and H2Lpy,NH and the
evaluation of their coordination behavior towards the Ga3+ ion. Herein, we also discuss
the molecular structures of the resulting Ga(III) complexes, which were assessed in
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solution and in the solid state based on multinuclear NMR studies and on Xꢀray
diffraction analysis, respectively. In parallel, the coordination behavior towards Ga3+ of
the precursor Schiff base ligands, H2Lpz*,C=N and H2Lpy,C=N, was also investigated. For
those complexes that were obtained as wellꢀidentified species, the synthesis of the
radioactive congeners was attempted using 67Ga and their in vitro stability was tested
under transchelation experiments with transferrin, and their biodistribution and
pharmacokinetics were evaluated in mice.
Results and Discussions
Synthesis of Ligands and Ga(III) complexes. The synthesis of the N4O2ꢀdonor ligands
was initiated with the introduction of the pyrazolylꢀ and pyridylꢀcontaining pendant
arms at the central nitrogen atom of the dien backbone (Scheme 1). This comprised the
synthesis of the previously reported compound (2ꢀ{(2ꢀtertꢀbutoxycarbonylaminoethyl)ꢀ
[2ꢀ(3,5ꢀdimethylpyrazolꢀ1ꢀyl)ꢀethyl]amino}ethyl) carbamic acid tertꢀbutyl ester (2),
which was obtained by the literature method.23 The pyridyl congener, i.e. tertꢀbutyl Nꢀ
[2ꢀ[2ꢀ(tertꢀbutoxycarbonylamino)ethylꢀ(2ꢀpyridylmethyl)amino]ethyl]carbamate
(4),
was obtained in similar way by Nꢀalkylation of N,N’ꢀbis(tertꢀ
a
butoxycarbonyl)diethylenetriamine (1) with 1ꢀbromomethylpyridine. Removal of the
BOC protecting groups of 2 and 4, under acid hydrolysis, afforded respectively
compounds 3 and 5 containing two terminal primary amines. Treatment of 3 and 5 with
salicylaldehyde in refluxing methanol, using a 1:2 molar ratio of the reagents, gave the
Schiffꢀbase ligands H2Lpz*,C=N and H2Lpy,C=N, respectively. The corresponding reduced
derivatives, H2Lpz*,NH and H2Lpy,NH, were synthesized by reaction of H2Lpz*,C=N and
H2Lpy,C=N with sodium borohydride in methanol. After appropriate workꢀup, these new
N4O2ꢀdonor ligands were obtained as pale yellow oils in moderate to high yield. Their
1
characterization was done by HRꢀESIꢀMS, IR, H and 13C NMR spectroscopy, which
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1
confirmed the formulation proposed for the compounds. In particular, the H NMR
spectra of H2Lpz*,C=N and H2Lpy,C=N in CDCl3 showed a downfield singlet at 8.05 ppm
1
due the imine N=CH–Ar proton. These imine proton resonances are absent in the H
NMR spectra of H2Lpz*,NH and H2Lpy,NH confirming the reduction of the imine function,
which is further corroborated by the presence in the spectra of both compounds of a
singlet at 3.89 ppm due to the protons of the new CH2 group.
Scheme 1. Synthesis of the ligands. i) O(BOC)2, THF, r.t., o.n.; ii) BrCH2CH2(3,5ꢀ
Me2pz) or 2ꢀBrCH2py , K2CO3, KI, THF, reflux, 24 h; iii) HCl/MeOH, r.t., 2 h; iv)
salicylaldehyde, MeOH, reflux, 4 h; v) NaBH4, MeOH, r.t., o.n.
As shown in Scheme 2, the attempts to synthesize the Ga(III) complexes with H2Lpz*,NH
and H2Lpy,NH were made by reaction with gallium nitrate in methanol in the presence of
sodium acetate, followed by precipitation with NaClO4. These reactions led to the
formation of [GaLpz*,NH](ClO4) and [GaLpy,NH](ClO4), which were isolated in the pure
form in low to moderate yield. These yields are justified by the purification processes
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needed to separate any free ligand and/or free gallium. Their characterization comprised
ESIꢀMS, C,H,N analysis, Xꢀray diffraction analysis, IR and 1D and 2D multinuclear
NMR.
Scheme 2. Synthesis of the gallium complexes. i) H2Lpz*,NH or H2Lpy,NH, NaCH3COO,
MeOH, r.t., o.n.; ii) NaClO4.
We have also investigated the capability of the imine derivatives to form gallium
complexes suitable for in vivo studies but the complexes were highly susceptible to
decomposition reactions originating from the wellꢀknown high reactivity of the imine
linkage. From these studies, the only isolated complex was [GaLpz*,OMe](ClO4)
coordinated by a hexadentate hemiaminal derivative as a result of the attack of one of
the imine groups of Lpz*,C=N by methanol. See Supporting Information for full details
and characterization of [GaLpz*,OMe](ClO4).
Solid State Molecular Structures. Crystals of [GaLpz*,NH](ClO4) and
[GaLpy,NH](ClO4) suitable for Xꢀray diffraction analysis were obtained from slow
evaporation of concentrated solutions of the compounds in methanol. For
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[GaLpz*,NH](ClO4), Xꢀray quality crystals were also grown from a concentrated solution
of the compound in chloroform. A summary of the crystal data, structure solution and
refinement parameters are given in Table 1. A selection of bond lengths and angles is
presented in Table 2.
Table 1: Crystallographic details for the Gallium Complexes
[GaLpz*,NH](ClO4).(MeOH)
[GaLpz*,NH](ClO4).(CHCl3)
[GaLpy,NH](ClO4).(MeOH)
empirical form.
fw
C26H37N5O7ClGa
636.78
C26H34N5O6Cl4Ga
724.10
C24H28N4O6ClGa
573.67
temp. (K)
cryst. system
space group
a (Å)
b (Å)
c (Å)
150(2)
monoclinic
P21/c
7.9018(3)
11.6269(4)
30.2610(11)
90
91.358(1)
90
150(2)
monoclinic
P21/c
14.2384(5)
12.5399(4)
17. 0528(6)
90
98.761(1)
90
150(2)
monoclinic
P21/n
22.5793(5)
10.5056(2)
23.2529(4)
90
117.545(1
90
α
β
γ
(deg)
(deg)
(deg)
V (Å3)
2779.40(17)
4,1.522
3009.22(18)
4,1.598
4890.57(18)
8,1.558
Z, Dcalcd (Mg/m3)
µ (mmꢀ1)
1.141
1.319
1.283
F(000)
1328
1488
2368
θ range (deg)
h,k,l range
reflns col./uniq.
T max./min.
S on F2
2.67ꢀ25.34
ꢀ9/9,ꢀ13/14,ꢀ36/30
13307/5077 [Rint=0.0440]
0.8945 /0.7714
1.072
2.59ꢀ25.35
ꢀ16/17,ꢀ15/11,ꢀ20/20
21810/5497 [Rint=0.0552] 72909/9277 [Rint=0.0444]
3.25ꢀ25.68
ꢀ27/19,ꢀ12/12,ꢀ24/28
0.9018 /0.7601
1.070
0.8210 /0.6551
1.046
0.0395
0.0942
0.0432
0.0252
0.0609
R1 (I > 2σ(I))
wR2 (all data)
0.1027
aDefinitions: R1 = ∑||Fo| ꢀ |Fc||/∑|Fo|, wR2 = [∑w(Fo ꢀ Fc2)2/∑w(Fo ) ]
2
2 2 1/2
Table 2. Selected Bond Lengths (Å) and Angles (º) for the Gallium Complexes
[GaLpz*,NH](ClO4).(MeOH) [GaLpz*,NH](ClO4).(CHCl3)
[GaLpy,NH](ClO4).(MeOH)
mol A
1.9021(12)
1.8800(12)
2.0900(15)
2.1329(15)
2.1203(15)
2.0941(14)
95.91(5)
90.74(5)
97.63(6)
85.13(5)
99.89(6)
mol B
Ga1ꢀO1
Ga1ꢀO2
Ga1ꢀN1
Ga1ꢀN2
Ga1ꢀN3
Ga1ꢀN4
O1ꢀGa1ꢀO2
O1ꢀGa1ꢀN1
O2ꢀGa1ꢀN1
O1ꢀGa1ꢀN4
O2ꢀGa1ꢀN4
N1ꢀGa1ꢀN4
O1ꢀGa1ꢀN3
O2ꢀGa1ꢀN3
1.9158(17)
1.8879(19)
2.127(2)
2.165(2)
2.073(2)
2.121(2)
94.12(8)
90.71(8)
90.77(9)
84.72(8)
96.05(8)
72.04(9)
172.77(9)
92.24(9)
1.886(2)
1.874(2)
2.121(3)
2.157(3)
2.116(3)
2.144(3)
97.64(10)
91.87(10)
90.42(10)
84.77(10)
97.15(9)
172.05(10)
171.32(10)
90.70(10)
1.9003(12)
1.8753(12)
2.0870(15)
2.1389(15)
2.1220(14)
2.0838(14)
95.40(5)
90.11(6)
99.21(6)
86.17(5)
98.72(6)
162.32(6) 161.95(6)
172.65(6) 172.58(6)
90.42(5)
91.32(5)
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N1ꢀGa1ꢀN3
N4ꢀGa1ꢀN3
O1ꢀGa1ꢀN2
O2ꢀGa1ꢀN2
N1ꢀGa1ꢀN2
N4ꢀGa1ꢀN2
N3ꢀGa1ꢀN2
92.63(9)
91.21(9)
93.64(8)
169.14(8)
81.54(9)
92.22(9)
80.53(9)
90.46(10)
91.84(10)
91.61(10)
168.20(10)
81.93(10)
90.95(9)
92.12(6)
90.12(6)
92.15(5)
91.93(6)
89.70(6)
91.15(5)
171.92(5) 173.25(5)
82.85(6)
80.14(6)
81.50(6)
82.40(6)
80.03(6)
82.06(6)
80.44(10)
The molecular structures of the cations of [GaLpz*,NH](ClO4) and [GaLpy,NH](ClO4) are
presented in Figures 1 and 2, respectively. Both compounds possess three chiral
coordinated nitrogen atoms. For [GaLpz*,NH](ClO4), the measured crystals contain a
unique enantiomeric form in the asymmetric unit, having S, R and R absolute
configurations at the N1, N2 and N3 nitrogen atoms, either when the crystals were
obtained from concentrated methanol or chloroform solutions of the compound. By
contrast, for [GaLpy,NH](ClO4) there are two independent molecules in the asymmetric
unit corresponding to two enantiomers with S, R, R and R, S, S absolute configurations
at the N1, N2 and N3 nitrogen atoms, respectively (Fig. 2).
Figure 1. ORTEP drawing at the 40% probability level for the [GaLpz*,NH]+ cation in
[GaLpz*,NH]ClO4. Hydrogen atoms are omitted for clarity with exception of those
attached to the coordinating aliphatic amine groups.
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Figure 2. ORTEP drawing at the 40% probability level for the two enantiomers of
[GaLpy,NH]+ cation in [GaLpy,NH]ClO4. Hydrogen atoms are omitted for clarity with
exception of those attached to the coordinating aliphatic amine groups.
The GaꢀO, GaꢀNpz and GaꢀNpy bond distances of [GaLpz*,NH](ClO4) and
[GaLpy,NH](ClO4) span in the ranges 1.874(2)ꢀ1.9158(17) Å, 2.121(2)ꢀ2.144(3) Å and
2.0838(14)ꢀ2.0941(14) Å, respectively. These values compare well with those reported
for other Ga(III) complexes containing phenoxide, pyrazolyl and pyridyl coordinating
groups.11,18,24,25 The GaꢀN bond distances of the coordinated pyrazolyl ring in
[GaLpz*,NH](ClO4), GaꢀN = 2.121(2) Å in crystals obtained from CHCl3 and GaꢀN =
2.144(3) Å in the crystals obtained from MeOH, are slightly longer than the GaꢀN bond
distances found for the coordinated pyridyl rings in the two enantiomeric forms that are
present in the measured crystal of [GaLpy,NH](ClO4) (GaꢀN = 2.0941(14) Å in molecule
A and GaꢀN = 2.0838(14) Å in molecule B). This trend reflects most probably the
poorer electronꢀreleasing properties of pyrazolyl, together with the greatest rigidity
inherent to the pyridylꢀcontaining fiveꢀmembered [GaN2C2] chelating ring in
comparison with the pyrazolylꢀcontaining sixꢀmembered [GaN3C2] chelating ring.
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Solution NMR structural analysis
Octahedral complexes of the type MA2B2CD with hexadentate chelators based on the
dien framework can exist as fac isomers, containing the three dien nitrogen atoms
coordinated in the same face of the octahedron, or as mer isomers where the same
nitrogen atoms occupy a meridional plane. [GaLpz*,NH]+ and [GaLpy,NH]+ cations can
therefore exist as fac and/or mer isomers in solution.
Compounds [GaLpz*,NH](ClO4) and [GaLpy,NH](ClO4) were studied by a variety of
multinuclear and bidimensional NMR techniques to identify the number of species in
solution and assign the coordination mode of the respective chelators in solution. The
assignment of the different diastereotopic methylenic protons of all species and
corresponding carbon atoms was achieved by means of COSY and HSQC spectra, and
in the case of [GaLpy,NH]+ also by TOCSY experiments (See Supporting Information).
The 1H and 13C spectra of [GaLpz*,NH]+ in acetonitrile solution revealed the presence of
a major species in solution, as can be checked in the 1H NMR spectrum that is shown in
Figure S3. The NOESY spectrum of such species (Figure 3) shows negative noe cross
peaks between each pair of diastereotopic protons, which denotes slow exchange
between enantiomers. This process also gives rise to indirect noe effects. To aid in the
assignment of the coordination geometry of the complex a series of selective noe 1D
spectra were also run (Figure S9). Based on these data, we concluded that the pyrazolyl
ring occupies an axial position of the octahedron, while the opposite axial position is
occupied by the NH group at 3.59 ppm (Figure 3). In agreement with this coordination
mode, the protons from the pyrazolyl methyl substituent appearing at 2.35 ppm are noeꢀ
correlated with a CH2 proton at 3.97 ppm from one of the sixꢀmembered rings
containing a phenoxide group. The NH proton at 3.65 ppm points downward and is only
correlated with a CH2 proton at 2.63 ppm from one of the fiveꢀmembered chelating ring
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containing axial and equatorially coordinated aliphatic amine groups. Furthermore, the
CH2 proton of the pyrazolyl arm at 4.22 ppm is closer to an axial proton at 2.73 ppm
from the same fiveꢀmembered chelating ring. On contrary, its geminal counterpart at
4.45 ppm has no noe correlations with any of the CH2 protons of the other fiveꢀ
membered chelating ring. In conclusion, the results of NOESY experiments indicated
that the structure of [GaLpz*,NH]+ in solution corresponds to the molecular structure that
was determined in the solid state (Fig. 1). The minor species may correspond to a merꢀ
N3dien isomer but it was not identified because the amount in solution is too small to
perform NMR studies.
Figure 3. a) Aliphatic region of the NOESY spectrum of [GaLpz*,NH]+ in CD3CN. A
slow exchange process between enantiomers is manifested by the presence of negative
noe cross peaks between each pair of diastereotopic protons (black peaks). This process
also gives rise to indirect noe effects that are present in this spectrum. b) Relevant NOE
enhancements used to confirm the facꢀN3dien coordination of GaLpz*,NH in CD3CN.
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Unlike [GaLpz*,NH](ClO4), the dissolution of [GaLpy,NH](ClO4) in acetonitrile gives
raise to three species detectable by NMR: two major species (A and B) and a minor
species (C) (Figure 4 and Scheme 3). A and B are coordination isomers and C is in
exchange with A. This exchange process is due most probably to the replacement of the
pyridyl arm by adventitious water.
1
Figure 4. Aromatic region of the H spectrum of [GaLpy,NH]+ in CD3CN showing the
presence of two major isomers A and B in a 1:0.6 ratio. Some of the peaks of the minor
species C are also identified.
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Scheme 3: [GaLpy,NH]+ species present in acetonitrile solution.
The chemical shifts of the coordinated NH groups (Figure 5 and Figure S13) are in
general very similar for the three species indicating closely related structures in
solution. The assignment of the diastereotopic methylenic protons in species A and B
was achieved by combining the results of several NMR experiments such as COSY
(Figure S10), TOCSY (Figure S11) and HSQC (Figure S12). For all species, the
coordination geometry around the gallium ion is octahedral, as suggested by the careful
analysis of the protonꢀproton correlations observed in the NOESY spectra (Figure S14).
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Figure 5. Relevant NOE enhancements used to confirm the merꢀN3dien coordination in
isomer A and the facꢀN3dien coordination in isomer B of [GaLpy,NH]+ in CD3CN.
In species A the pyridyl arm is trans to a phenolate oxygen and together with the three
amine nitrogen donor atoms define two adjacent faces of an octahedral (Scheme 3).
This corresponds to merꢀN3dien configuration because one phenolate oxygen is cis to
the pyridyl arm and the other one is trans. The two fiveꢀmembered chelating rings
containing the aliphatic amines are puckered in a similar way but one of NH protons
(NHa) lies on the same side as the pyridyl group while the other is pointing downward
in the opposite direction (NHb) (Figure 5). Thus, NHa at 4.16 ppm has a long range noe
with the CH2 protons of the pyridyl arm resonating at 4.39 ppm while NHb at 4.27ppm
does not correlate with these protons. The NHa amine proton has also a very weak cross
peak with the pyridyl Ho doublet at 8.97 ppm. This ortho proton shows another
correlation with a CH2 proton at 3.36 ppm from the phenoxideꢀcontaining sixꢀ
membered ring in the opposite side of the molecule.
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The isomer B possesses also an octahedral geometry but the pyridyl is now trans to the
NHa group (Figure 5). The other two amine nitrogen atoms and NHa belong to the same
face of the octahedron. The NHb proton at 3.79 ppm points downwards relative to the
pyridyl arm and is directed towards NHa at 4.16 ppm. Thus, NHb has a long range noe
with NHa; an axial CH2 proton at 3.80 ppm from one of the phenoxide–containing sixꢀ
membered chelating ring correlates with the ortho proton from the pyridyl ring at 8.92
ppm (Figures 5 and S14). All these findings are consistent with the solid state molecular
structures depicted in Figure 2. We call this isomer facꢀN3dien because both phenoxide
oxygens are cis to the pyridyl ring.
The NOESY spectrum of [GaLpy,NH]+ (Fig. S14) in CD3CN also shows negative cross
peaks as a result of a slow exchange process that interconverts species A and C (black
peaks). A variable temperature study (Fig. S15) also confirmed the presence of this
process. The ortho proton of the pyridyl ring in species C resonates at 8.22 ppm being
considerably upfield shifted when compared with the same proton (δ = 8.92 ppm) in
species A, and approaching the chemical shift of the ortho pyridyl proton in free
H2Lpy,NH
(δ = 8.45 ppm). These data are consistent with the dissociation of the pyridyl
group in A, which must be accompanied by the coordination of a solvent molecule.
Rapid exchange in solution hampered its identification by NMR but we believe that
adventitious water is probably replacing the pyridine arm instead of acetonitrile itself
(Scheme 3). H2O is a harder donor ligand than CH3CN and, therefore, is expected to be
1
a better ligand towards the oxophilic Ga3+ ion. Indeed, the H NMR spectrum of
[GaLpy,NH]+ in D2O shows that B and C are the predominant species in aqueous
solutions (Figure 6). The NOESY spectrum of [GaLpy,NH]+ (Figure S17) in D2O also
shows negative cross peaks as a result of a slow exchange process that interconverts
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species C and A (black peaks). The variable temperature study (Fig. S18) also confirms
the presence of this process. The assignment of the diastereotopic CH2 protons (Figure
S16), based on a variety of 2D experiments (Figures S19ꢀS21) associated with a fine
analysis of the NOESY, confirmed that the structure of B is maintained in D2O solution.
Due to deuteration of NH protons and the scarce number of noe contacts observed for
C, it was impossible to confirm that the ligand arrangement in C match that in A.
However, the downfield shift of some CH2 protons in C (Figure S16) is also observed in
A, which seems to indicate that the ligand arrangement in these interconverting species
is similar. The exception is the presence of a free pyridyl group in C, as indicated by
ortho protons resonating at 8.19 ppm and without no long range noe contacts (Figure
S17b).
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1
Figure 6. Aromatic region of the H NMR spectra of [GaLpy,NH]+ in D2O showing the
presence of two major isomers B and C in a 1:0.7 ratio. Minor peaks are from isomer A.
Despite the presence of two coordination isomers in solution, the 71Ga NMR spectrum
of [GaLpy,NH]+ in acetonitrile displays a single but relatively broad resonance (ꢁν1/2
~
5700 Hz) centred at 102 ppm, which can be certainly accounted by presence of the same
donorꢀatom set in isomers A and B. The broadness of the 71Ga signal is even higher in
the 71Ga spectra of [GaLpz*,NH]+ (
δ = 53 ppm, ꢁν1/2 ~12000 Hz) indicating a larger
asymmetry of the coordination environment around trivalent gallium in this complex
(Figure S22).26
In summary, the larger trans influence of a phenolate oxygen when compared to an
aliphatic amine can certainly account that the pyridyl arm is labile in species A but not
in species B.27,28 Such trans influence may also justify the coordination of both
phenoxide groups in cis position relatively to the pyrazolyl arm in [GaLpz*,NH](ClO4),
observed either in solution or in the solid state.
Radiolabelling and Biological Studies. For H2Lpz*,NH and H2Lpy,NH that were able to
coordinate in a intact form the Ga3+ ion, we have pursued with the study of the synthesis
of the respective gallium complexes at carrier free level using 67Ga. The synthesis of the
radiocomplexes was attempted by treating solutions of 67GaCl3 in acetate buffer (pH =
5) with ethanolic solutions of the ligands. For a final H2Lpy,NH concentration of 6.25x10ꢀ
5 M and after heating at 85 ºC for 15 min, the HPLC analysis of the reaction mixture has
shown the presence of a single radioactive peak (tR = 18.3 min). This peak corresponds
to [67GaLpy,NH]+ since its retention time is wellꢀmatched with the one of cold (i.e. non
radioactive) [GaLpy,NH]+ (tR = 18.2 min) (Fig. 7a). [67GaLpy,NH]+ was obtained in almost
quantitative radiochemical yield (RCY) (RCY > 95 %), as confirmed by the HPLC
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analysis and thin layer chromatography (TLC) that discarded the presence of
radiochemical impurities corresponding to hydrolyzed forms of gallium. By contrast,
H2Lpz*,NH provided only very low amounts (< 25 %) of the desired radioactive
compound ([67GaLpz*,NH]+) even when a final H2Lpz*,NH concentration as high as ca. 1
x10ꢀ3 M was used (Fig. 7b). These differences are not explained by the weaker basicity
of the pyrazolyl arm (pKa = 2.5 for protonated pyrazole) if compared with the pyridine
arm (pKa = 5.2 for protonated pyridine),29 as this implies a larger percentage of nonꢀ
protonated pyrazole available for complexation to the metal.
Figure 7. HPLC chromatograms of: a) [GaLpy,NH](ClO4) (UV detection) and reaction
mixture of the radiolabeling of H2Lpy,NH with 67GaCl3 (γ detection); b)
[GaLpz*,NH](ClO4) (UV detection) and reaction mixture of the radiolabeling of
H2Lpz*,NH with 67GaCl3 (γ detection).
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To have a further insight on the interest of H2Lpy,NH as a new ligand framework for the
design of gallium radiopharmaceuticals, we have investigated the in vitro stability of
[67GaLpy,NH]+ in challenge experiments with human apoꢀtransferrin and we have studied
its biodistribution and in vivo stability in mice.
The ionic radius of octahedral Ga(III) (0.62 Å) is comparable to that of highꢀspin
octahedral Fe(III) (0.645 Å). Both these metal ions are hard Lewis acids, which present
a strong affinity for hard Lewis bases, in particular to oxygen and nitrogen donors. For
these reasons, Ga(III) has a very high affinity for transferrin, which is a iron transport
protein with a relatively high concentration (ca. 2.5 mg/mL) in human blood and
presenting two homologous regions for iron coordination. Each region can coordinate
independently one Fe3+ ion (or Ga3+), along with a carbonate or bicarbonate anion per
metallic ion. The high affinity of Ga(III) for transferrin explains that radiocomplexes of
Ga are usually tested in challenge experiments against this protein, particularly when
evaluating new chelator systems,11,13 as in the present case. These challenge
experiments are useful to predict the in vivo kinetic inertness of the complexes towards
transchelation reactions. [67GaLpy,NH]+ was incubated with a large excess (ca. 100:1) of
apoꢀtransferrin in the presence of bicarbonate at 37 ºC and the reaction mixture was
analysed by HPLC at different time points (Fig. S23). No transchelation was observed
even after 48 h of incubation, which attests for the high in vitro stability of
[67GaLpy,NH]+ and for the suitability of H2Lpy,NH to stabilize Ga3+ under the condition
involved in the preparation of radiopharmaceuticals. Under the same HPLC analytical
conditions, 67GaCl3 (tR = 2.3 min) and Tf-67Ga (tR = 4.3 min) (Fig. S24) have shown
retention times significantly different from that of [67GaLpy,NH]+, which further
validated the results from the challenge experiments.
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To ascertain the biokinetics and in vivo stability of [67GaLpy,NH]+ , biodistribution
studies were carried out in CDꢀ1 mice at 1 h and 4 h after intravenous administration.
Total radioactivity excretion was also assessed at both time points. The most relevant
data from these studies are summarized in the graphic of Figure 8. [67GaLpy,NH]+ shows
a fast blood clearance and a moderately fast overall rate of excretion. Noticeably,
[67GaLpy,NH + showed a faster blood clearance than 67Ga complexes with pyridylꢀ
]
containing acyclic chelators of the H2dedpa type (see Chart 1) which have been recently
introduced by Orvig et al. According to these authors, the retention of [67Ga-dedpa]+ in
the blood compartment could be explained by a strong association with serum proteins
due to the secondary amine groups of the chelator.11 Clearly, this is not the case for
[67GaLpy,NH]+ despite the presence of two secondary amines in the structure of the
complex.
45
1h
4h
40
35
30
25
20
15
10
5
0
Blood
Liver
Intestine Spleen
Heart
Lung
Kidney
Bone
Muscle Stomach
Brain
Excretion
Figure 8. Biodistribution results of [67GaLpy,NH]+ in female CDꢀ1 mice showing organ
and tissue uptake (expressed in %I.D./g) and overall excretion at 1 h and 4 h after i.v.
administration.
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In general, [67GaLpy,NH]+ presented a pronounced clearance from most organs and
tissues with the exception of those involved in the excretion of the compound, such as
kidney, liver and intestine. In the elimination of the complex, there is a significant
contribution of the hepatobiliary excretion pathway despite the moderate hydrophilicity
of [67GaLpy,NH]+ (Log Do/w = ꢀ0.53 ± 0.04). The involvement of this excretory route
slightly delays the overall radioactivity excretion when compared with GaꢀNOTA and
with tris(hydroxypyridinone) based Ga compounds.13,30,31 Comparably to these Ga
chelates, [67GaLpy,NH]+ also shows a negligible bone uptake (< 0.1 % ID/g at 4 h p.i.),
which indicates a high in vivo stability for this complex since it is well known that free
Ga3+ tends to accumulate in the bone.32 The in vivo stability of [67GaLpy,NH]+ was
further corroborated by radioꢀHPLC analysis of serum and urine from CDꢀ1 mice
injected with this radiocomplex. The obtained radiochromatograms revealed uniquely
the presence of intact [67GaLpy,NH]+ (Fig. S25). Altogether, the biodistribution profile of
[67GaLpy,NH]+ is consistent with a high in vivo stability.
Conclusions
New chelators, H2Lpz*,NH and H2Lpy,NH, were designed based on the dien backbone by
introducing additional pyrazolyl and pyridyl coordinating units at the central nitrogen
atom and by functionalization of the dien terminal amines with phenol groups. The
coordination behaviour of these chelators towards the Ga3+ ion was evaluated having in
mind to assess their suitability for the design of gallium radiopharmaceuticals. For nonꢀ
radioactive gallium, these studies allowed the synthesis and full characterization by
different spectroscopic techniques and Xꢀray diffraction analysis of [GaLpz*NH]+ and
[GaLpy,NH +
]
that contain N4O2ꢀhexadentate chelators. These complexes have a
remarkably different behaviour in solution, as showed by a variety of multinuclear and
bidimensional NMR techniques. These studies demonstrated that [GaLpy,NH]+ exists in
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solution as a mixture of coordination isomers, while a predominant isomer was detected
for [GaLpz*,NH]+. At carrier free level, i.e. using radiogallium (67Ga), notorious
differences were also observed for the behaviour of H2Lpz*,NH and H2Lpy,NH
[67GaLpy,NH +
.
]
was synthesized in almost quantitative radiochemical yield using
relatively low ligand concentrations (ca. 5x10ꢀ5 M), while [67GaL2]+ was hardly formed
even using a 50ꢀfold higher concentration of H2Lpz*,NH. For comparative purposes, the
imine congeners H2Lpz*,C=N and H2Lpy,C=N were also studied but the high reactivity of
the imine bonds did not allow the synthesis of Ga(III) complexes with relevance for
radiopharmaceutical research.
[67GaLpy,NH]+ has shown a high in vivo stability and a favourable biodistribution profile
in mice. Thus, it can be considered a promising building block for the design of gallium
radiopharmaceuticals. It may have relevance for the design of perfusion agents, e.g. for
cardiac imaging, as different substituents can be introduced at the secondary amines,
pyridyl and phenolate rings to tune the lipophilicity of the resulting Ga(III) complexes.
However, the H2Lpy,NH backbone can also be explored to design bifunctional chelators
for targetꢀspecific radiopharmaceuticals because the pyridyl ring is easily replaceable by
picolinic acid derivatives presenting carboxylic groups for linkage to targeting
biomolecules.
Altogether, our findings highlighted that the combination of dien framework with Nꢀ
heterocyclic and phenol coordinating groups can provide N4O2ꢀdonor chelators suitable
to stabilize Ga(III) complexes for in vivo studies. The encouraging results obtained for
H2Lpy,NH justify further studies aiming at a better understanding of the structural factors
(e.g. nature of the Nꢀheterocyclic group and spacer length) that determine the
performance of this type of compounds as chelators for the design of
radiopharmaceuticals.
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Experimental Section
Materials and Methods: Unless stated otherwise, the synthesis and workꢀup of the
ligands and complexes were performed under air. The compounds N,N’ꢀbis(tertꢀ
butoxycarbonyl)diethylenetriamine (1)33 and (2ꢀ{(2ꢀtertꢀbutoxycarbonylaminoethyl)ꢀ[2ꢀ
(3,5ꢀdimethylpyrazolꢀ1ꢀyl)ꢀethyl]amino}ethyl) carbamic acid tertꢀbutyl ester (2)23 were
prepared according to published methods. 67GaCl3 was prepared from the readily
available [67Ga]ꢀGallium Citrate (Mallinckrodt) using SEPꢀPAK SI cartridges (Waters)
1
as previously reported.34 The H, 13C, 71Ga and 15N NMR spectra of the Ga(III)
complexes were recorded at room temperature on a Bruker Avance III spectrometer
1
13
operating at 500, 125, 152 and 50 MHz, respectively. The H and C NMR spectra of
organic compounds and ligands were recorded on a Varian Unity operating at 300 and
1
75 MHz, respectively. Chemical shifts of H and 13C spectra (δ, ppm) are reported
relative to the residual solvent peaks relative to SiMe4. Spectra in D2O are referenced to
15
TSP. For 71Ga and N spectra, external references were used: Ga(NO3)3 in D2O (δ = 0
ppm) and neat CH3NO2 (δ = 381.7 ppm relative to liquid NH3), respectively. Peak
assignments were achieved via the use of COSY, TOCSY, HSQC, NOESY and
selective NOEs at 500 MHz. IR spectra were recorded in nujol using KBr mulls or as
KBr pellets on a Bruker, Tensor 27 spectrometer. Electrospray ionisation mass
spectrometry (ESIꢀMS) was performed at Centro de Ciências e Tecnologias Nucleares
(C2TN) on a QITMS instrument in positive ion mode. High resolutionꢀmass
spectrometry (HRMS), using ESIꢀFIAꢀTOF, was carried out as a service by the
University of Santiago de Compostela. For the organic compounds, thinꢀlayer
chromatography (TLC) was performed on Merck silica gel 60 F254 plates and column
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chromatography using silica gel 60 (Merck). RPꢀTLC of the cold Ga and 67Ga
complexes was done using TCLꢀsheets Alugram RPꢀ18W/UV (MachareyꢀNagel) and
MeOH/0.9% NaCl (1:1) as eluent. HPLC analysis of the cold Ga and 67Ga complexes
was performed on a Perkin–Elmer LC pump 200 coupled to a LC 290 tunable UVꢀVis
detector and to a Berthold LBꢀ507A radiometric detector, using an analytical Machereyꢀ
Nagel C18 reversedꢀphase column (Nucleosil 100–10, 250 x 4 mm) with a flow rate of
1 mL minꢀ1 and gradient elution with a mixture of aqueous 0.9% NaCl (A) and ethanol
(B); Method: 0ꢀ10 min, 100% A; 10ꢀ15 min, 100ꢀ0% A; 15ꢀ25 min, 0% A; 25ꢀ26 min,
0ꢀ100% A; 26ꢀ30 min, 100% A.
Synthesis of N'-(2-aminoethyl)-N'-[2-(3,5-dimethylpyrazol-1-yl)ethyl]ethane-1,2-
diamine (3): To solution of compound 2 (826 mg, 1.94 mmol) in MeOH (25 mL), at
0ºC, were added 6 mL of 12 M HCl. The reaction mixture was left to stir at room
temperature for 24 h. Then, the solvent was evaporated under vacuum, the pH was
adjusted to pH=14 by addition of 40 % aqueous NaOH and the mixture extracted with
dichloromethane (3x20 mL). The organic phase was dried over MgSO4, filtered and the
filtrate was dried under vacuum to afford the title compound as a pale yellow oil. Yield:
341 mg (78 %).
1
HRMS (C11H23N5): m/z Calcd for [M + H]+ 226.2026; Found 226.2018. H
,
NMR (293K, 300 MHz, CD3OD): δH 2.17 (3H, s, CH3), 2.22 (3H, s, CH3), 2.51 (4H, tr,
CH2), 2.68 (4H, t, CH2), 2.82 (2H, t, CH2), 4.00 (2H, tr, CH2), 5.75 (1H, s, H(4)ꢀpz). 13C
NMR (293K, 75 MHz, CDCl3): δc 10.84 (CH3), 13.11 (CH3), 38.96 (CH2), 38.96 (CH2),
46.54 (CH2), 53.68 (CH2), 55.45 (CH2), 104.78 (C(4)ꢀpz), 138.58 (C(3/5)ꢀpz), 146.85
(C(3/5)ꢀpz).
Synthesis
of
tert-butyl
N-[2-[2-(tert-butoxycarbonylamino)ethyl-(2-
pyridylmethyl)amino]ethyl]carbamate (4): In a 100 mL flask, 2 (700 mg, 2.26
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mmol), 1ꢀbromomethylꢀpyridine (628 mg, 2.48 mmol), K2CO3 (685 mg, 4.96 mmol)
and KI (41 mg, 0.25 mmol)) were mixed in 30 mL of dried THF. The mixture was
refluxed overnight. The solution was filtered and the solvent was evaporated under
reduced pressure to afford the final product. Yield: 786 mg (99%).
1
HRMS (C20H34N4O4): m/z Calcd for [M + H]+ 395.2653; Found 395.2650. H
,
NMR (293K, 300 MHz, CDCl3): δH 1.42 (18H, s, 2ꢀC(CH3)3, 2.62 (4H, t, 2ꢀCH2), 3.15
(4H, t, 2ꢀCH2), 3.74 (2H, s, CH2), 5.51 (2H, b, 2ꢀNH), 7.15 (1H, t, py), 7.29 (1H, d, py),
7.62 (1H, t, py), 8.52 (1H, d, py). 13C NMR (293K, 75 MHz, CDCl3): δc 28.32 (CH3),
38.34 (CH2), 54.03 (CH2), 59.80 (CH2), 78.71 (C(CH3)3), 121.98 (py), 122.83 (py),
136.37 (py), 148.99 (py), 156.17 (py), 159.37 (C=O).
Synthesis of N'-(2-aminoethyl)-N'-(2-pyridylmethyl)ethane-1,2-diamine (5): 25 mL
of MeOH were added to compound 4 (1.0 g, 2.5 mmol) in a 50 mL flask. The solution
was placed in an ice bath and 12 M HCl (6 mL) was added. The mixture was stirred at
room temperature for 24 h. The solvent was evaporated under reduced pressure and to
the resulting residue was added 40% aqueous NaOH until pH=14. The compound was
then extracted with CHCl3 (3x20 mL), dried with MgSO4 and the organic solvent
evaporated under vacuum. Yield: 466 mg (96%).
1
HRMS (C10H18N4): m/z Calcd for [M + H]+, 195.1604; Found 195.1601. H
NMR (293K, 300 MHz, CDCl3): δH 2.56 (4H, t, 2ꢀCH2), 2.73 (4H, t, 2ꢀCH2), 3.72 (2H,
s, CH2), 7.13 (1H, t, py), 7.37 (1H, d, py), 7.62 (1H, t, py), 8.49 (1H, d, py). 13C NMR
(293K, 75 MHz, CDCl3): δc 39.58 (CH2), 57.59 (CH2), 60.78 (CH2), 122.00 (py),
122.89 (py), 136.44 (py), 149.04 (py), 159.83 (py).
Synthesis of H2Lpz*,C=N (2-[(E)-2-[2-(3,5-dimethylpyrazol-1-yl)ethyl-[2-[(E)-(2-
hydroxyphenyl)methyleneamino]ethyl]amino]ethyliminomethyl]phenol):
A
solution of compound 3 (296 mg, 1.31 mmol) and salicylaldehyde (321 mg, 2.63 mmol)
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in MeOH (15 mL) was refluxed under N2 for 4 h, and then evaporated to dryness under
reduced pressure. The residue was extracted several times with diethyl ether (3x25 mL)
and the ether fractions were filtrated to remove an insoluble orange material. The filtrate
was dried under vacuum giving a pale yellow oil, which was formulated as H2Lpz*,C=N
Yield: 256 mg (45%).
.
HRMS (C25H31N5O2): m/z Calcd for [M + H]+ 434.2551; Found 434.2567. IR
,
(KBr, ν/cm–1) 3428w, 3019m; 2952m; 2901m, 2253w, (N=C, sharp) 1634vs, 1582m
1553w, 1498m; 1461m; 1280s; 1216s; 1152w; 1117w; 1072w; 1041w; 900s; 760s;
1
669sh,m; 650mw; 556w; 464w. H NMR (293K, 300 MHz, CDCl3) δH 2.03 (3H, s,
CH3), 2.19 (3H, s, CH3), 2.83 (4H, t, 2ꢀCH2), 2.96 (2H, tr, CH2), 3.54 (4H, tr, 2ꢀCH2),
3.89 (2H, tr, CH2), 5.70 (1H, s, H(4)ꢀpz), 6.78 (2H, tr, Ph), 6.90 (2H, d, Ph), 7.00 (2H,
d, Ph), 7.26 7.00 (2H, tr, Ph), 8.05 (2H, s, CH=N), 13.38 (2H, br, OH); 13C NMR
(293K, 75 MHz, CDCl3) δc 10.84 (CH3), 13.49 (CH3), 47.08 (CH2), 54.93 (CH2), 55.73
(CH2), 57.84 (CH2), 104.77 (C(4)ꢀpz), 116.91 (Ph), 118.46 (Ph), 118.66 (Ph), 131.27
(Ph), 132.11 (Ph), 139.35 (C3/5ꢀpz), 147.32 (C3/5ꢀpz), 161.16 (Ph), 165.95 CH=N).
Synthesis
of
H2Lpz*,NH
(2-[[2-[2-(3,5-dimethylpyrazol-1-yl)ethyl-[2-[(2-
hydroxyphenyl)methylamino]ethyl]amino]ethylamino]methyl]phenol): A solution
of compound 3 (390 mg, 1.73 mmol) and salicylaldehyde (423 mg, 3.46 mmol) in
MeOH (20 mL) was refluxed under N2 for 4 h. After cooling to room temperature, solid
NaBH4 (200 mg, 5.29 mmol) was added to the reaction mixture and the resulting clear
solution was stirred overnight at room temperature. The solvent was evaporated under
reduced pressure, the oily residue was redissolved in dichloromethane and then washed
with a saturated NaHCO3 aqueous solution. The organic phase was separated, the
solvent removed under vacuum and the crude was purified by silicaꢀgel column
chromatography using CH2Cl2/MeOH/ NH4OH (90/30/2)) as the eluent. Removal of the
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