Rhenium(v) oxocomplexes with novel pyrazolyl-based N4- and N3S-donor chelators

Article abstract of DOI:10.1039/b611034g

The novel pyrazolyl-based ligands 3,5-Me₂pz(CH₂) ₂NH(CH₂)₂NH(CH₂)₂NH ₂ (1) and pz*(CH₂)₂NH-Gly-CH ₂STrit (pz* = pz (8), 3,5-Me₂

Full text of DOI:10.1039/b611034g

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www.rsc.org/dalton | Dalton Transactions
Rhenium(V) oxocomplexes with novel pyrazolyl-based N4- and N3S-donor
chelators
Carolina Moura, Rute F. V´ıtor, Leonor Maria, Anto´nio Paulo, Isabel C. Santos and Isabel Santos*
Received 1st August 2006, Accepted 26th September 2006
First published as an Advance Article on the web 10th October 2006
DOI: 10.1039/b611034g
The novel pyrazolyl-based ligands 3,5-Me2pz(CH2)2NH(CH2)2NH(CH2)2NH2 (1) and
pz*(CH2)2NH-Gly-CH2STrit (pz* = pz (8), 3,5-Me2pz (9), 4-(EtOOC)CH2-3,5-Me2pz (10))
were synthesized, and their suitability to stabilize Re(V) oxocomplexes was evaluated using
different starting materials, namely (NBu4)[ReOCl4], [ReOCl3(PPh3)2] and trans-[ReO2(py)4]Cl.
Compound 1 reacts with trans-[ReO2(py)4]Cl yielding the cationic compound
[ReO(OMe){3,5-Me2pz(CH2)2N(CH2)2NH(CH2)2NH2}](BPh4) (11) in a low isolated yield. In contrast,
the neutral complexes [ReO{pz*(CH2)2NH-Gly-CH2S}] (pz* = pz (12), 3,5-Me2pz (13),
4-(EtOOCCH2)-3,5-Me2pz (14)) were synthesized almost quantitatively by reacting [ReOCl3(PPh3)2] or
(NBu4)[ReOCl4] with the trityl-protected chelators 8–10. The X-ray diffraction analysis of 11 and 13
confirmed the tetradentate coordination mode of the respective ancillary ligands. In 11 the
monoanionic chelator coordinates to the metal through four nitrogen atoms, while in 13 the chelator is
trianionic, coordinating to the metal through three nitrogens and one sulfur atom. Solution NMR
studies of 12–14, including two-dimensional NMR techniques (1H COSY and 1H/13C HSQC),
confirmed that the N3S coordination mode of the chelators is retained in solution. Unlike 11,
complexes 12–14 may be considered relevant in the development of radiopharmaceuticals, as further
corroborated by the synthesis of the congener [99mTcO{pz(CH2)2-NH-Gly-CH2S}] (12a). This
radioactive compound was obtained from 99mTcO4 in aqueous medium, in almost quantitative yield
−
and with high specific activity and radiochemical purity.
Introduction
The development of radiopharmaceuticals is one of the major
driving forces for the coordination and organometallic chemistry
of technetium and rhenium since 99mTc is the most used radionu-
clide in diagnostic nuclear medicine, while Re has two b-emitter
radionuclides (186/188Re) with excellent physical decay properties
for therapeutic applications.1–4 Re compounds are also usually
considered adequate surrogates of the 99mTc complexes.
We have shown that tridentate pyrazolyl-diamine ligands are
efficient bifunctional chelators towards the fac-[M(CO)3]+ (M =
Re, 99mTc) moiety (Chart 1), having relevance in the design of
organometallic Tc and Re compounds for radiopharmaceuti-
cal applications.5,6 Within this organometallic approach, these
chelators have been already applied for labelling small tumor-
seeking peptides, with encouraging results in terms of biological
profile and in vitro/in vivo stability of the bioactive organometallic
complexes.7,8 The favourable features of pyrazolyl-diamine ligands
led us to evaluate the possibility of preparing pyrazolyl-based
Chart 1 Schematic drawing of reported M(I) tricarbonyl and expected
M(V) complexes (M = Re, 99mTc) anchored by pyrazolyl-based chelators.
tetradentate chelators suitable for the synthesis of Re(V) and Tc(V)
oxocomplexes, another class of compounds with a prominent
role in the radiopharmaceutical chemistry of these d-transition
right combination of donor atoms. Tetradentate N4 ligands, of the
elements.1,2 The use of Re(V) or Tc(V) oxocomplexes for designing
tetraamine type, are among the best bifunctional chelators for the
radiopharmaceuticals requires the stabilisation of trans-[MO2]+ or
trans-[MO2]+ core, while a variety of N3S-donor ligands have been
[MO]3+ cores (M = Re, Tc) with bifunctional chelators having the
applied with success to stabilise the [MO]3+ core.2,9,10 Therefore
the combination of a pyrazolyl group with an aliphatic triamine
or with a mercaptoacetylglycine unit would provide potentially
tetradentate chelators of the N4 and N3S type, which were expected
Departamento de Qu´ımica, ITN, Estrada Nacional 10, 2686-953, Sacave´m
Codex, Portugal. E-mail: isantos@itn.pt
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to stabilise the trans-[MO2]+ and the [MO]3+ (M = Re, Tc) cores,
respectively (Chart 1). The presence of an aromatic pyrazolyl ring
in the framework of these chelators would also confer some rigidity
to the complexes and allows an easy coupling of biologically
relevant molecules to the metal center.11–14
(H2N(CH2)2N(Boc)(CH2)2NH(Boc))16 (Scheme 1). By exploring
this synthetic route we have also taken into account that Boc-
protected pyrazolyl-triamine would be useful for the introduction
of a pendant arm at the non-protected secondary amine for further
coupling to bioactive molecules.
Herein, we describe a series of novel tetradentate pyrazolyl-
based ligands, 3,5-Me2pz(CH2)2NH(CH2)2NH(CH2)2NH2 (1) and
pz*(CH2)2NH-Gly-CH2STrit (pz* = pz (8), 3,5-Me2pz (9), 4-
(EtOOCCH2)-3,5-Me2pz (10)), and report on their coordination
chemistry towards trans-[ReO2]+and [ReO]3+ cores. To gain insight
into the suitability of these novel chelators for radiopharmaceuti-
cal development, some preliminary studies at the no-carrier added
level (99mTc) have been performed and will be also reported in this
work.
The N3S-donor ligands, 8–10, were synthesized by a mul-
tistep process which involved a final amide coupling reac-
tion between N-(2-aminoethyl)pyrazole precursors, 5–7, and
N-hydroxysuccinimidyl 2-[(triphenylmethylthio)methylcarbonyl-
amino]ethanoate11,17 in the presence of DIPEA and in refluxing
acetonitrile (Scheme 2). The compounds pz(CH2)2NH2 (5)18
and 3,5-Me2pz(CH2)2NH2 (6)19 were prepared by reacting the
corresponding bromoethylpyrazoles with potassium phthalimide,
followed by hydrazinolysis of the phthalimide intermediates 2 and
3. Compound 4-(EtOOCCH2)-3,5-Me2pz(CH2)2NH2 (7) has been
synthesized via a Mitsunobu reaction (Scheme 2)20 by treatment of
4-(EtOOCCH2)-3,5-Me2pz(CH2)2OH6 with phthalimide, followed
by reaction with hydrazine.
Results and discussion
Synthesis and characterization of the pyrazolyl-based chelators
Compounds 8–10 have been purified by column chromatogra-
phy and were used in the synthesis of the Re(V) oxocomplexes
without previous detritylation of the thiol groups, as discussed
below. Their formulation was supported by elemental analysis,
IR, 1H and 13C NMR spectroscopy.
The chelator 3,5-Me2pz(CH2)2NH(CH2)2NH(CH2)2NH2 (1) has
been prepared by two different synthetic approaches (Scheme 1).
One of these approaches consisted of reacting N-(2-p-
toluenesulfonylethyl)-3,5-dimethylpyrazole15 with a large excess
of diethylenetriamine (1 : 20) (Scheme 1). This reaction leads to
1
chelator 1, a pale-yellow oil which has been characterized by H
and 13C NMR spectroscopy, but also to a side product formed due
to alkylation of the secondary amine, as can be seen in Scheme 1.
To avoid the formation of the side product, chelator 1 was
prepared alternatively by reacting N-(2-p-toluenesulfonylethyl)-
3,5-dimethylpyrazole with the Boc protected diethylenetriamine
Synthesis and spectroscopic characterization of the Re(V)
oxocomplexes
The reaction of trans-[ReO2(py)4]Cl with 1 (1 : 1 molar ratio)
in refluxing methanol yields a dark green solution which, after
Scheme 1 Synthesis of the ligand 3,5-Me2pz(CH2)2NH(CH2)2NH(CH2)2NH2 (1).
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Scheme 2 Synthesis of pz*(CH2)2NH-Gly-STrit ligands.
1
removal of the solvent, was analysed by H NMR spectroscopy.
probably the formation of the Re(V) monoxocomplex, 11, and/or
the deprotonation of the aliphatic amine of chelator 1. We have
carried out the studies under aqueous conditions, aiming to avoid
the formation of the oxo-methoxyde complex and to disfavour the
deprotonation of 1. The precursor trans-[ReO2(py)4]Cl reacts with
1 (1 : 1 molar ratio) in H2O affording a clear orange solution, after
heating at 80 ◦C for 1 h. The solvent was removed under vacuum
The 1H NMR analysis has shown the presence of a major species
which clearly had the pyrazolyl-triamine ligand coordinated. This
assumption was based on the chemical shift (d = 6.36 ppm) of
the H(4) proton of the pyrazolyl ring, considerably downfield
shifted in comparison with the same proton in compound 1 (d =
5.83 ppm). Moreover, a complex set of multiplets, between 2.73
and 4.57 ppm, could also be easily assigned to the presence
of diastereotopic methylenic protons as a consequence of the
coordination of the amine groups of 1 to the metal. Expecting
to have cationic complexes, the recrystallization of this reaction
mixture has been attempted in methanol, using tetraphenylborate
as counterion. After standing for several days at room temperature,
a few dark green crystals formed which were of a suitable quality
for single crystal X-ray diffraction analysis. As discussed below,
this structural study confirmed the formation of [ReO(OMe){3,5-
Me2pz(CH2)2N(CH2)2NH(CH2)2NH2}](BPh4) (11) (Scheme 3): a
cationic Re(V) oxo-methoxyde complex anchored by a monoan-
ionic pyrazolyl-based N4-donor ligand. Unfortunately, the crys-
talline material was only sufficient to perform the X-ray structural
study, and 11 has not been studied by other analytical techniques.
The presence of the pyrazolyl ring, certainly a better r + p
donor than secondary or primary aliphatic amines, facilitates most
1
and the crude product analysed by H NMR spectroscopy. The
spectrum obtained in D2O has shown a pattern similar to the
one obtained when the reaction was run in methanol: a highfield
shifted resonance at 6.11 ppm due to the H(4) proton of the
pyrazolyl ring and a complex set of CH2 multiplets spanning
between 2.66 and 4.06 ppm. We must also mention that the IR
spectrum of the crude product did not show any band in the range
−1
=
900–1000 cm , where m(Re O) stretching bands usually appear,
displaying instead a medium intense band at 800 cm−1 which can
be assigned to mas(O Re O).21 These data might indicate the pres-
ence of a Re(V) trans-dioxocomplex anchored by 1. However, some
unreacted trans-[ReO2(py)4]Cl was still present and, therefore, one
=
=
=
=
could not unequivocally associate the mas(O Re O) band to the
novel species formed with chelator 1. Several attempts have been
made to purify this compound, namely precipitation with sodium
tetraphenylborate in aqueous solution or in organic medium. All
Scheme 3 Synthesis of a Re(V) oxocomplex with chelator 1.
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the attempts were unsuccessful and the presence of a Re(V) trans-
dioxocomplex anchored by 1 could not be confirmed.
starting material (NBu4)[ReOCl4] allowed the preparation of 14 in
a high isolated yield (80%), even at room temperature.
To evaluate the coordination behaviour of the tritylated lig-
ands pz*(CH2)2NH-Gly-CH2STrit (pz* = pz (8) 3,5-Me2pz (9))
[ReOCl3(PPh3)2] was used as the Re(V) starting material. In
refluxing methanol and using triethylamine as a deprotonating
agent, the reactions proceeded smoothly, affording almost quan-
titatively (97–98%) the neutral complexes [ReO{pz*(CH2)2-NH-
Gly-CH2S}] (pz* = pz (12), 3,5-Me2pz (13)) (Scheme 4). These
results showed that the cleavage of the C–S bond of the trityl
protecting group is a very efficient process, certainly promoted by
the Lewis acidity of the Re(V).12,22
Compounds 12–14 are carmine or orange microcrystalline
solids which are soluble in polar organic solvents, like
dichloromethane, chloroform, methanol or acetonitrile, but in-
soluble in water. These Re(V) oxocomplexes are quite stable in the
solid state and in solution, even in the presence of air or moisture,
as checked by 1H NMR and HPLC analysis. The characterization
of 12–14 has been performed by elemental analysis, IR and NMR
spectroscopy (1H, 13C, 1H COSY and 1H/13C HSQC) and, in the
case of 13, also by X-ray diffraction analysis.
=
The IR spectra of 12–14 display intense m(Re O) stretching
bands in the range 971–984 cm−1.21 There are also one or two
strong bands, spanning between 1628–1655 cm−1,12 which were
assigned to the m(CO) stretching bands of the two amide groups
present in the N3S chelators.
The 1H NMR spectra of 12–14 display a complex array of
resonances due to the methylenic protons of the linear backbone
of the respective pz*(CH2)2NH-Gly-CH2S chelators. These reso-
nances are downfield shifted in comparison with the ones due to
the corresponding protons of the free N3S ligands (Table 1). A
characteristic feature of all these spectra is the presence of two
sets of doublets between 3.98 and 5.44 ppm belonging to spin
systems of the AB type, as exemplified for complex 12 in fig-
=
ure 1. These doublets were assigned to the endo (syn to the Re O
=
group) and to the exo protons (anti to the Re O group) of the
mercaptoacetylglycine unit (Table 1), by comparison with the 1H
NMR data reported in the literature for other Re(V) complexes
bearing this coordinating motif.12,22 For each pair of endo/exo
methylenic protons, the resonance attributed to the endo proton
was the one displaced towards highfield.
Scheme 4 Synthesis of Re(V) oxocomplexes with the pz*-(CH2)2NH–
Gly–CH2S ligands.
In the case of 4-(EtOOCCH2)-3,5-Me2pz(CH2)2NH-Gly-
CH2STrit (10) the reaction with [ReOCl3(PPh3)2] in refluxing
methanolwas less favourable. The complex[ReO{4-(EtOOCCH2)-
3,5Me2pz(CH2)2-NH-Gly-CH2S}] (14) was also formed, but the
major part of the protected ligand did not react after 24 h of
reflux. As shown in Scheme 4, the use of the more reactive Re(V)
For complexes 13 and 14 in CDCl3, the resonances due to
the CH2 protons of the ethylenic bridge linking the mercap-
toacetyl unit and the pyrazolyl ring appear as three relatively
broad multiplets, spanning between 3.53 and 4.74 ppm (Table 1).
These protons should originate four multiplets, due to their
Table 1 1H and 13C NMR for the methylenic backbone of ligands 8–10 and complexes 12–14
Compound
1H NMR dH (ppm)c
13C NMR dC (ppm)
pz-Gly-STrit (8)a
Ha, 4.20; Hb, 3.63; Hc, 3.50; Hd, 3.14
Ha, 4.00; Hb, 3.58; Hc, 3.50; Hd, 3.13
Cc, 43.4; Cd, 35.5; C O, 168.5, 168.7
Ca, 46.8; Cb, 39.3; Cc, 43.2; Cd, 35.5; C O, 168.3, 168.5
=
3,5-Me2pz-Gly-STrit (9)a
=
4-(EtOOC)CH2-3,5-Me2-pz-Gly-STrit (10)a Ha, 4.07; Hb, 3.54; Hc, 3.54; Hd, 3.08
Ca, 46.8; Cb, 39.3; Cc, 43.2; Cd, 38.0; C O, 168.4, 168.5
Ha, 5.02/4.49; Hb, 4.60/3.13; Hc, 4.64/4.31; Hd, Ca, 53.7; Cb, 46.8; Cc, 55.9.2; Cd, 35.5; C O, 188.6,
=
[ReO(pz-Gly-S)] (12)b
=
4.06/3.83
189.9
[ReO(3,5-Me2pz-Gly-S)] (13)a
Ha, 4.50/3.53d; Hb, 4.67/3.53d; Hc, 5.44/4.35;
Hd, 4.05/3.98
Ca, 50.6; Cb, 42.7; Cc, 55.9; Cd, 39.1; C O, 190.4, 191.2
=
Ha, 4.54/3.47d; Hb, 4.74/3.47d; Hc, 5.43/4.35;
Hd, 4.03/3.91
Ca, 50.8; Cb, 42.5; Cc, 55.9; Cd, 39.1; C O, 190.4, 191.2
=
[ReO{4-(EtOOC)CH2-3,5-Me2-pz-Gly-S}]
(14)b
a Spectra run in CDCl3. b Spectra run in dmso-d6. c Each pair is relative to the Hexo/Hendo protons attached at the same carbon. d There is occasional overlap
for these protons.
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Table 2 Crystal data and structure refinement for compounds 11 and 13
Compound
11
13
Formula
M
Crystal system
Color
C36H45BN5O2Re
776.78
Monoclinic
Green
C22H30N8O7Re2S2
955.02
Triclinic
Carmine
Crystal size/mm
0.33 × 0.30 × 0.24
0.20 × 0.10 × 0.08
¯
P1
Space group
P21/n
˚
a/A
12.101(2)
14.3993(15)
19.975(2)
90
96.285(13)
90
3459.6(9)
4
293(2)
9.4388(2)
10.3213(3)
14.9377(3)
89.0390(10)
80.1750(10)
89.2970(10)
1433.6(3)
2
˚
b/A
˚
c/A
a/◦
b/◦
c /◦
3
˚
V/A
Z
T/K
130(2)
2.212
8.638
Dc/gcm−3
1.491
3.55
l (Mo Ka)/mm−1
Reflections collected
Independent reflections
Final R indices [I > 2r(I)]
R1
wR2
R ´ındices (all data)
R1
wR2
GOF
7102
17158
6767 [Rint = 0.0551] 12499 [Rint = 0.0225]
Fig. 1 1H NMR spectrum of complex 12 (range 5.0–3.0 ppm) in dmso-d6
(* residual water from the solvent).
0.0663
0.0910
0.0396
0.0927
diastereotopic character, but there is the occasional overlapping
of the two resonances associated with each exo proton from
the two methylenic groups, as confirmed by two-dimensional
NMR techniques (1H COSY and 1H/13C HSQC). The 1H NMR
spectrum of 12 in dmso-d6 display the expected four multiplets for
these protons, as shown in Fig. 1.
0.1474
0.1127
1.047
0.0586
0.1073
1.003
◦
˚
Table 3 Selected bond lengths (A) and angles ( ) for the cation of
compound 11
The CH and CH3 protons of the pyrazolyl ring are downfield
shifted (Dd = 0.3–1.6 ppm) compared to the corresponding
protons in the free ligands, confirming that these rings are
coordinated to the [ReO]3+ core. In the 13C NMR spectra of
12–14 all the resonances are downfield shifted relative to the
corresponding resonances in the free chelators. This trend is more
pronounced for the carbons that are close to the coordinating
groups, as is the case of the amide carbons (Dd = 22–23 ppm)
or the glycine a carbon (Dd = 12–13 ppm). In summary, the
NMR data obtained for 12–14 are compatible with the formation
of diamagnetic Re(V) oxocomplexes anchored by trianionic and
tetradentate N3S-donor ligands, as found in the solid state for one
complex of this family (13).
Re–O(1)
Re–N(1)
Re–N(4)
1.708(6)
2.090(8)
2.101(8)
157.8(3)
92.8(3)
86.4(3)
84.4(3)
89.6(4)
106.4(3)
159.6(3)
130.7(7)
115.1(9)
Re–O(2)
Re–N(3)
Re–N(5)
1.948(6)
1.913(8)
2.299(8)
98.7(3)
83.4(3)
94.5(3)
74.5(3)
167.7(3)
83.0(3)
78.9(3)
114.2(7)
O(1)–Re–O(2)
O(1)–Re–N(4)
O(2)–Re–N(1)
O(2)–Re–N(4)
N(1)–Re–N(3)
N(1)–Re–N(5)
N(3)–Re–N(5)
C(2)–N(3)–Re
C(2)–N(3)–C(3)
O(1)–Re–N(1)
O(1)–Re–N(5)
O(2)–Re–N(3)
O(2)–Re–N(5)
N(1)–Re–N(4)
N(3)–Re–N(4)
N(4)–Re–N(5)
C(3)–N(3)–Re
X-Ray crystallography
X-Ray quality crystals of complexes [ReO(OMe){3,5-
Me2pz(CH2)2N(CH2)2NH(CH2)2NH2}](BPh4) (11) and [ReO{3,5-
Me2pz(CH2)2-NH-Gly-CH2S}] (13) were obtained by slow
evaporation of methanol and acetonitrile solutions, respectively.
Crystal data for 11 and 13 are summarized in Table 2.
An ORTEP view of the cation of 11 is shown in Fig. 2, and a
selection of bond distances and angles for this complex is presented
in Table 3.
Compound 13 crystallized with two independent and chemically
different molecules per asymmetric unit cell. The most striking
difference between these two molecules is the absence (molecule
Fig. 2 ORTEP view of the cation of compound 11. Vibrational ellipsoids
A) or presence (molecule B) of a coordinated H2O ligand in a trans
are drawn at the 30% probability level.
3+
=
position relatively to the [Re O] core. Selected bond distances
and angles for molecules A and B of complex 13 are given in
Table 4. ORTEP views for each of these molecules are presented
in Fig. 3 and 4.
For complex 11 and for molecule B of compound 13 the coor-
dination geometry around the metal is approximately octahedral,
whereas the structure of molecule A of 13 can be considered as
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◦
˚
Table 4 Selected bond lengths (A) and angles ( ) for 13
Molecule A
Re(1)–O(1)
Re(1)–N(3)
1.690(4)
1.997(4)
Re(1)–N(1)
Re(1)–N(4)
2.111(4)
1.981(4)
Re(1)–S(1)
2.2847(13)
100.74(18)
107.04(18)
88.98(16)
88.27(12)
132.98(12)
O(1)–Re(1)–N(1)
O(1)–Re(1)–N(4)
N(1)–Re(1)–N(3)
N(1)–Re(1)–S(1)
N(3)–Re(1)–S(1)
O(1)–Re(1)–N(3)
O(1)–Re(1)–S(1)
N(1)–Re(1)–N(4)
N(3)–Re(1)–N(4)
N(4)–Re(1)–S(1)
113.44(18)
113.18(14)
152.19(17)
78.93(18)
82.15(14)
Molecule B
Re(2)–O(4)
Re(2)–N(6)
1.682(3)
1.999(4)
Re(2)–O(5)
Re(2)–N(7)
2.306(3)
2.040(3)
Re(2)–N(9)
2.143(8)
Re(2)–S(2)
2.3379(11)
104.11(17)
92.24(16)
86.31(14)
77.25(14)
79.22(15)
83.05(11)
153.31(11)
O(4)–Re(2)–O(5)
O(4)–Re(2)–N(7)
O(4)–Re(2)–S(2)
O(5)–Re(2)–N(7)
O(5)–Re(2)–S(2)
N(6)–Re(2)–N(9)
N(7)–Re(2)–N(9)
N(9)–Re(2)–S(2)
169.8(15)
101.95(16)
101.63(11)
78.30(14)
80.80(9)
162.16(15)
90.58(15)
100.84(11)
O(4)–Re(2)–N(6)
O(4)–Re(2)–N(9)
O(5)–Re(2)–N(6)
O(5)–Re(2)–N(9)
N(6)–Re(2)–N(7)
N(6)–Re(2)–S(2)
N(7)–Re(2)–S(2)
Fig. 4 ORTEP view of molecule B of 13. Vibrational ellipsoids are drawn
at the 30% probability level.
a methoxyde ligand coordinated in a trans-position relative to the
oxo group (O(1)–Re(1)–O(2) angle of 157.8(3)◦).
In all the structures the Re atom is out of the distorted
basal plane, towards the oxo group. As expected, this is more
˚
pronounced for molecule A of 13, with a 0.663(2) A displacement
of the metal from the [N(1), N(3), N(4), S(1)] plane. The Re–O
˚
bond distance in 11 (1.708(6) A) appears at the upper limit usually
found for multiple bound oxo ligands in Re(V) oxocomplexes,21
being longer than the same distance in the two independent
˚
molecules of compound 13 (molecule A: Re(1)–O(1), 1.690(4) A;
˚
molecule B: Re(2)–O(4), 1.682(3) A). This difference is certainly
justified by the competition of the methoxide group with the oxo
ligand in p-bonding with the metal, as evidenced by the relatively
˚
short Re–O(2) bond distance (1.948(6) A) found for compound
11.24 By contrast, the loosely bound water ligand in molecule B
25
˚
of 13 (Re(2)–O(5), 2.306(3) A) is not expected to compete in an
extensive way with the oxo-ligand.
Concerning the coordination of the tetradentate ancillary
ligands the Re–Npz, Re–Namine, Re–Namide and Re–S bond distances
can be considered normal, taking into account the values reported
for the same bond distances in other Re(V) complexes with
pyrazolyl containing ligands,26,27 polyamines28,29 and MAG3 (mer-
captoacetyltriglycine) chelators.12,21,30,31 In particular, the short Re–
Fig. 3 ORTEP view of molecule A of 13. Vibrational ellipsoids are drawn
at the 30% probability level.
distorted square pyramidal. In terms of trigonality index s, the
value found for the latter is 0.32 (s = 0 for a regular square pyramid,
s = 1 for a regular trigonal bipyramid).23 In all the structures,
the ancillary ligands are coordinated in a tetradentate way, with
the respective donor atoms occupying the equatorial plane or the
basal square plane. The tetradentate pyrazolyl-triamine ligand in
complex 11 is monoanionic, due to deprotonation of the secondary
amine adjacent to the pyrazolyl ring. The 3,5-Me2pz(CH2)2-NH-
Gly-CH2S ligand is trianionic in both independent molecules of
13. The oxo ligands occupy the axial (11 or molecule B of 13) or
apical positions (molecule A of 13). In the case of complex 11,
the coordination environment around the metal is completed by
˚
N(3) bond distance of 1.913(8) A in compound 11 is consistent
with a certain double character for the coordination of the
deprotonated and almost planar sp2-hybridized nitrogen atom.28
This bond distance is considerably shorter than the Re–Namine bond
˚
distances in 11 (2.090(8)–2.299(8) A), being more comparable to
˚
the Re–Namido bond distances in 13 (1.981(4)–2.040 A).
Labelling studies: syntheses and in vitro evaluation of
[
99mTcO{pz(CH2)2-NH-Gly-CH2S}] (12a)
The stability of the complexes [ReO{pz*(CH2)2-NH-Gly-CH2S}]
(pz* = pz (12), 3,5-Me2pz (13), 4-(EtOOCCH2)-3,5-Me2pz (14)),
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both in the solid state and in solution, together with their excellent
isolated yields (80–98%), even using trityl-protected ligands,
prompted us to pursue with the studies at the no-carrier added level
Concluding remarks
A novel class of tetradentate chelators containing a pyrazolyl unit
have been synthesized and their coordination capability towards
the trans-[Re(O)2]+ and [M(O)]3+ cores (M Re, 99mTc) studied. Us-
ing the pyrazolyl-triamine ligand (1) no Re(V) complex containing
a trans-[ReO2]+unit could be isolated. The unique compound iso-
lated with 1 was the mono-oxocomplex [ReO(OMe){3,5-Me2pz-
(CH2)2N(CH2)2NH(CH2)2NH2}](BPh4) (11). This compound has
no relevance in radiopharmaceutical research, since the trans-
[ReO(OMe)]2+ unit is not achievable under the aqueous conditions
required in the preparation of radiopharmaceuticals.
(
99mTc). In order to gain insight into the possibility of preparing
=
99mTc oxocomplexes with this class of chelators, we have focused
on pz(CH2)2NH-Gly-CH2STrit (8). Immediately before use, an
ethanolic solution of 8 was heated (100 ◦C, 30 min) in the presence
of aqueous 0.1 N HCl, to remove the trityl-protecting group. The
complex [99mTcO{pz(CH2)2-NH-Gly-CH2S}] (12a) was prepared
by reacting [99mTcO4]− with tin(II) chloride, Na/K tartrate, and an
aliquot of the trityl-deprotected 8 (Scheme 5).
By contrast, a series of trianionic and tetradentate ligands,
combining a pyrazolyl ring with a mercaptoacetylglycine unit,
allowed the synthesis and full characterization of well defined
complexes, [ReO{pz*-(CH2)2-NH-Gly-CH2S}] (pz* = pz (12),
3,5-Me2pz (13), 4-(EtOOCCH2)-3,5Me2-pz (14)), which may have
some relevance in radiopharmaceutical development. These com-
plexes are remarkably stable, either in the solid state or in solution,
and can be prepared almost quantitatively using S-protected
ligands. The potential relevance of this class of complexes for
the design of radiopharmaceuticals was further demonstrated by
the synthesis of [99mTcO{pz-(CH2)2-NH-Gly-CH2S}] (12a), which
was obtained in almost quantitative yield and with high specific
activity. The neutral and moderately lipophilic M(V) (M = Re,
Tc) complexes anchored on the pz*-(CH2)2-NH-Gly-CH2S ligands
must be suitable for labelling small biomolecules, for which free
diffusion through the cell membrane can be a crucial issue.
Preferentially, these small biomolecules can be attached to the
framework of the pz*-(CH2)2-NH-Gly-CH2S chelators through
derivatization of the 4-position of the pyrazolyl ring, as exemplified
herein with the synthesis of complex 14 which already contains a
CH2COOEt ester function useful for coupling biomolecules.
Scheme 5 Synthesis of a 99mTc(V) oxocomplex with the pz-(CH2)2NH–
Gly–CH2S ligand.
Complex 12a was obtained with high radiochemical yield
(> 90%), at room temperature and using ligand concentrations
in the 10−5 M range. The TLC analysis of 12a, using methanol
as eluent, has shown the presence of negligible amounts of
reduced hydrolyzed technetium (< 5%). In this chromatographic
system, complex 12a shows Rf = 0.7, as well as its Re congener
[ReO{pz(CH2)2-NH-Gly-CH2S}] (12) which was fully character-
ized, as discussed above. The identity of complex 12a (tR
=
14.63 min) was further authenticated by HPLC comparison with
complex 12 (tR = 14.00 min) (Fig. 5). Typically, HPLC analysis
of the preparations of 12a has shown that more than 95% of the
eluted radioactivity was due to complex 12a.
Experimental
General procedures
The synthesis of the ligands and complexes were carried under
a nitrogen atmosphere, using standard Schlenk techniques and
dry solvents, while the work-up procedures were performed under
air. The compounds N-(2-p-toluenesulfonylethyl-3,5-dimethyl-
pyrazole,15 N-(2-bromoethyl)pyrazole,32 N-(2-bromoethyl)-3,5-
dimethylpyrazole,32
N-(2-hydroxyethyl)-3,5-dimethyl-4-(ethyl-
ethanoate)pyrazole6 and N-hydroxysuccinimidyl 2-[(triphenyl-
methylthio)methylcarbonylamino]ethanoate11,17 were prepared
according to published methods. The starting materials trans-
[ReO2(py)4]Cl,33 [ReOCl3(PPh3)2]34 and [NBu4][ReOCl4]35 were
prepared by the literature methods. Na[99mTcO4] was eluted from
a commercial 99Mo/99mTc generator, using 0.9% saline.
1H and 13C NMR spectra were recorded on a Varian Unity
300 MHz spectrometer; 1H and 13C chemical shifts are given in ppm
and were referenced with the residual solvent resonances relative
to SiMe4. IR spectra were recorded as KBr pellets on a Bruker,
Tensor 27 spectrometer. C, H and N analyses were performed on
an EA 110 CE Instruments automatic analyser.
Fig.
5
HPLC chromatograms of co-injected [ReO{pz-(CH2)2NH-
Gly-CH2S}] (12) (UV detection, tR = 14.00 min) and [99mTcO{pz-(CH2)2-
NH-Gly-CH2S}] (12a) (c detection, tR = 14.63 min).
Complex 12a is moderately lipophilic (log Po/w (12a) = + 0.73
0.03), and displays a high in vitro stability in the presence of a
huge excess of glutathione (10–20 mM), since HPLC analysis has
shown that no trans-chelation occurred, at least for a period of
2 h at 37 ◦C. Glutathione is a tripeptide, present in relatively high
concentrations in the blood stream, with a well known affinity for
the [99mTcO]3+ core.
Thin layer chromatography (TLC) was done using plates
from Merck (silica gel 60 F254). Column chromatography was
performed in silica gel 60 (Merck). HPLC analysis was performed
5636 | Dalton Trans., 2006, 5630–5640
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on a Perkin-Elmer LC pump 200 coupled to a LC 290 tunable
UV/Vis detector and to a Berthold LB-507A radiometric detector,
using a Macherey-Nagel C18 reversed-phase column (Nucleosil
10 lm, 250 × 4 mm) and a gradient of aqueous 0.1% CF3COOH
(A) and acetonitrile (B) with a flow rate of 1.0 mL min−1. Method:
0–3 min, 100% A; 3–18 min, 100% → 0% A; 18–22 min, 0% A;
22–25 min 0%→ 100% A; 25–30 min, 100% A.
stirring at room temperature for 2 h, the solvent was removed
under vacuum and the residue redissolved in distilled water.
After adjustment to pH = 11 with 40% NaOH, the water was
removed under vacuum giving a residue which, after extraction
with methanol and upon evaporation of the solvent, afforded
compound 1. Yield: 38 mg (0.17 mmol, 31%).
1H NMR (300 MHz, CDCl3): dH 2.14 (s, 3H, CH3-pz), 2.18
(s, 3 H, CH3-pz), 2.67 (m, 6H, CH2), 2.77 (t, 2 H, CH2), 2.97
(t, 2 H, CH2,); 4.01 (t, 2 H, CH2), 5.72 (s, 1 H, H(4)-pz). 13C
NMR (75.373 MHz, CDCl3): dC 11.0 (CH3-pz), 13.4 (CH3-pz),
41.3 (CH2), 48.1 (CH2), 48.8 (CH2), 49.1 (CH2), 51.6 (CH2), 104.8
(C(4)-pz), 139.1 (C(3,5)-pz), 147.4 (C(3,5)-pz).
Syntheses of the ligands
3,5-Me2pz(CH2)2NH(CH2)2NH(CH2)2NH2 (1). Method 1:
A solution of N-(2-p-toluenesulfonylethyl-3,5-dimethylpyrazole
(1.75 g, 6 mmol) in THF (5 mL) was added dropwise, at
room temperature, to an aqueous solution of diethylenetriamine
(13 mL, 0.12 mol) and NaOH (250 mg, 6.25 mmol). After
complete addition, the mixture was refluxed for 4 h and extracted
with dichloromethane, after cooling to room temperature. After
concentration under vacuum, the organic extract was applied on
the top of a silica-gel column which was eluted with CH2Cl2–
MeOH, starting from 0 to 100% MeOH. Compound 1 was
obtained as a pale yellow oil upon removal of the solvents from
the collected methanol fractions. Yield: 634 mg (2.82 mmol, 48%).
Method 2(i), synthesis of H2N(CH2)2N(Boc)(CH2)2NH(Boc):
This compound was prepared by a method different from the one
described in the literature,16 starting from diethylenetriamine. A
solution of ethyl trifluoroacetate (1.46 g, 10. 3 mmol) in CH2Cl2
(12 mL) was added dropwise to a solution of diethylenetriamine
(1.06, 10.3 mmol) in the same solvent (12 mL), while keeping the
temperature at 0 ◦C. After complete addition, the mixture was
stirred for 2 h at 0 ◦C and then for 2 h at room temperature. Re-
moval of the solvent under vacuum yielded a yellow oil which was
formulated as H2N(CH2)2NH(CH2)2NH(COCF3) and used with-
out further purification. This compound was dissolved in CH2Cl2
(20 mL) and the resulting solution was cooled to −10 ◦C. Di-tert-
butyl dicarbonate (4.5 g, 20.6 mmol) dissolved in CH2Cl2 (20 mL)
was added dropwise to the cooled solution, and then the reaction
mixture was stirred for 24 h at room temperature. After evapo-
ration of the solvent, (Boc)NH(CH2)2N(Boc)(CH2)2NH(COCF3)
was obtained in the form of a dense oil, which was dissolved
in a mixture of methanol (280 mL) and distilled water (12 mL).
Then, K2CO3 (55.28 g, 0.4 mmol) was added and the mixture
refluxed for 2 h. After filtration, the solvent from the supernatant
was removed under vacuum, the residue was redissolved in
distilled water and the pH adjusted to pH = 13 with 40%
NaOH. Extraction with CHCl3, followed by removal of the
solvent from the organic phase, gave a yellow oil corresponding
to H2N(CH2)2N(Boc)(CH2)2NH(Boc). Yield: 2.05 g (7.6 mmol,
74%).
N-(2-Phthalimideethyl)pyrazole (2). A mixture of potassium
phthalimide (6.446 g, 34.8 mmol) and N-(2-bromoethyl)pyrazole
(2.06 g, 11.7 mmol) in acetonitrile was refluxed, under nitrogen,
for 3 d. After filtration, the solvent was evaporated under vacuum,
affording compound 2 as a microcrystalline white solid. Yield:
2.505 g (10.4 mmol, 89%).
1H NMR (300 MHz, CDCl3) dH 4.09 (2H, t, CH2), 4.44 (2H, t,
CH2), 6.19 (1H, t, H(4)-pz), 7.69 (1H, d, H(3/5)-pz), 7.70 (1H, d,
H(3/5)-pz), 7.76 (2H, m, Ph), 7.80 (2H, m).
N-(2-Phthalimideethyl)-3,5-dimethylpyrazole (3). Compound
3 was prepared as above described for 2, starting from
4.694 g (23.0 mmol) of N-(2-bromoethyl)-3,5-dimethylpyrazole
and 18.821 g (69.0 mmol) of potassium phthalimide, but its
purification was done by column chromatography (CH2Cl2–
MeOH (98 : 2)). Yield: 2.216 g (8.23 mmol, 56%)
1H NMR (300 MHz, CDCl3): dH 2.05 (3H, s, CH3-pz); 2.19
(3H, s, CH3-pz); 4.01 (2H, t, CH2); 4.25 (2H, t, CH2); 5.74
(1H, s, H(4)-pz); 7.72 (2H, m, Ph); 7.80 (2H, m, Ph). Rf (silica-gel,
CH2Cl2–MeOH (98 : 2)) = 0.30.
N-(2-Phthalimideethyl)-4-(methylpropionate)-3,5-dimethylpyra-
zole (4). N-(2-Hydroxyethyl)-3,5-dimethyl-4-(ethylethanoate)-
pyrazole (500 mg, 2.70 mmol) and phthalimide (516 mg,
3.51 mmol) were dissolved in dry THF (12 mL). Next, diethyl
azodicarboxylate (582 lL, 3.51 mmol) and PPh3 (921 mg,
3.51 mmol) were added and the mixture was allowed to stir
overnight at room temperature. The resulting red solution was
purified by silica gel column chromatography, by elution of a first
column with CH2Cl2–MeOH (95 : 5) and elution of a second
one with ethyl acetate–n-hexane (80 : 20)). Compound 4 was
recovered as a white powder, after removal of the solvent from the
collected fractions. Yield: 558 mg (1.57 mmol, 58%).
1H NMR (300 MHz, CDCl3): dH 1.13 (3H, t, CH3), 1.97 (3H, s,
CH3-pz), 2.10 (CH3-pz), 3.19 (2H, s, CH2), 3.91 (2H, t, CH2), 4.00
(2H, q, CH2), 4.17 (2H, m, CH2), 7.66 (2H, m, Ph), 7.77 (2H, m,
Ph). 13C NMR (75.373 MHz, CDCl3): dC 9.4 (CH3(Et)), 11.6 (CH3-
pz), 14.1 (CH3-pz), 29.8 (CH2), 37.5 (CH2), 46.1 (CH2), 60.6 (CH2),
109.5 (C(4)-pz), 123.2 (Cphthalimide), 133.9 (Cphthalimide), 137.0 (C(3/5)-
pz), 147.0 (C(3/5)-pz), 167.7 (COphthalimide), 171.4 (CO (Et)). Rf
(silica gel, ethyl acetate–n-hexane (80 : 20)) = 0.50.
(ii) Synthesis of 1: N-(2-p-toluenesulfonylethyl-3,5-dimethyl-
pyrazole (167 mg, 0.55 mmol) in THF (5 mL) was
added dropwise to an aqueous solution (10 mL) of
H2N(CH2)2N(Boc)(CH2)2NH(Boc) (162 mg, 0.55 mmol) and
NaOH (44 mg, 1.1 mmol). The mixture was refluxed for 24 h and,
after cooling to room temperature, the solution was extracted
with CHCl3. The organic extract was purified by column chro-
matography (CH2Cl2 (0–100%)–MeOH). A pale yellow oil was
recovered from the collected methanol fractions and formulated as
3,5-Me2pz(CH)2NH(CH2)2N(Boc)(CH2)2NH(Boc). To a solution
of this compound in CH2Cl2 was added TFA (250 lL). After
N-(2-Aminoethyl)pyrazole (5) and N-(2-aminoethyl)-3,5-
dimethylpyrazole (6). To a solution of 2 (1.013 g, 4.20 mmol)
or 3 (1.041 g, 3.86 mmol) in methanol were added, respectively,
2.20 mL or 2.00 mL of hydrazine monohydrate. The mixtures
were refluxed under nitrogen for 3 h, resulting in the formation
of a white precipitate of phthalhydrazide. After cooling to room
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temperature, concentrated HCl (2, 9.47 mL; 3, 8.70 mL) was
added, leading to the formation of more white precipitate. After
filtration and adjustment to pH = 9 by addition of 3M NaOH, the
supernatant was extracted with CHCl3 (3 × 20 mL). The organic
extracts were dried over MgSO4, filtered and the solvent removed
under vacuum to afford yellow oils which were formulated as
compounds 5 and 6. Yield: 5, 289 mg (2.60 mmol, 62%); 6, 340 mg
(2.44 mmol, 63%).
(CH-Trit), 139.5 (C(3/5)-pz); 143.8 (C-Trit); 148.0 (C(3/5)-pz);
168.3 (CO), 168.5 (CO). Rf (silica gel, CH2Cl2–MeOH (95 : 5)) =
0.30.
4-(EtOOC)CH2-3,5-Me2pz(CH2)2NH-Gly-CH2STrit
(10).
Compound 10 is a yellow oil which has been obtained as reported
above for 8 and 9, starting from 103 mg (0.45 mmol) of 7. Yield:
142 mg (0.24 mmol, 41%).
Found C, 63.1; H, 7.7; N, 9.2; S. C34H38N4O4S requires C, 64.6;
1
Compound 5, H NMR (300 MHz, CDCl3): dH 3.06 (2H, m,
1
H, 6.1; N, 8.7%. IR: mmax/cm−1 1670 (CO). H NMR (300 MHz,
CH2); 4.11 (2H, t, CH2); 6.18 (1H, t, H(4)-pz); 7.36 (1H, d, H(3,5)-
pz); 7.45 (1H, d, H(3/5)-pz).
CDCl3): dH 1.22 (3H, d, J = 7.2 Hz, CH3), 2.11 (3H, s, CH3-pz),
2.12 (3H, s, CH3-pz), 3.08 (2H, s, SCH2), 3.28 (2H, s, pzCH2COO),
3.54 (2H + 2H, m, CH2), 4.07 (2H + 2H, m, CH2), 6.74 (1H, br,
NH), 6.92 (1H, br, NH), 7.15–7.41 (15H, m, Ph-Trit). 13C NMR
(75.373 MHz, CDCl3): dC 9.3 (CH3), 11.6 (pz-CH3), 14.0 (pz-CH3),
29.31 (CH2), 35.4 (CH2), 39.1 (CH2), 42.9 (CH2), 46.9 (CH2), 60.6
(CH2), 109.0 (C(4)-pz), 127.3 (CH-Trit), 128.6 (CH-Trit), 129.2
(CH-Trit), 137.5 (C(3/5)-pz); 143.6 (C-Trit), 146.7 (C(3/5)-pz),
168.40 (CO), 168.50 (CO), 171.4 (CO). Rf (silica gel, CH2Cl2–
MeOH (95 : 5)) = 0.30.
1
Compound 6, H NMR (300 MHz, CDCl3): dH 2.17 (3H, s,
CH3-pz); 2.20 (3H, s, CH3-pz); 3.07 (2H, m, CH2); 3.98 (2H, t,
CH2); 5.76 (1H, s, H(4)-pz).
N-(2-Aminoethyl)-4-(methylpropionate)-3,5-dimethylpyrazole (7).
Compound 7 was prepared and purified in the same way as that
described above for 5 and 6, starting from 194 mg (0.55 mmol) of
4 and running the reaction in ethanol. Yield: 103 mg (0.46 mmol,
84%).
1H NMR (300 MHz, CDCl3): dH 1.21 (3H, t, CH3); 2.16 (3H, s,
CH3-pz); 2.18 (CH3-pz); 3.07 (2H, m, CH2); 3.43 (2H, s, CH2);
3.97 (2H, t, CH2); 4.16 (2H, q, CH2). 13C NMR (75.373 MHz,
CD3OD): dC 9.9 (CH3), 10.0 (CH3), 14.5 (CH3), 28.7 (CH2); 39.3
(CH2); 46.6 (CH2); 52.9 (CH2); 114.7 (C(4)-pz), 126.5 (C(3/5)-pz),
134.2 (C(3/5)-pz); 171.7 (CO).
Syntheses of the rhenium complexes
[ReO(OMe){3,5-Mepz(CH2)2N(CH2)2NH(CH2)2NH2}](BPh4)
(11). To a solution of trans-[ReO2(py)4]Cl (98 mg, 0.17 mmol)
in methanol (15 mL) was added 1 (43 mg, 0.19 mmol) dissolved
in the minimum volume of the same solvent and the mixture was
refluxed for 4 h, affording a dark green solution. Recrystallization
of the crude product in methanol and in the presence of NaBPh4
gave a few crystals of 11, which has been characterized only by
X-ray diffraction analysis.
pz(CH2)2NH-Gly-CH2STrit (8). The precursor N-hydroxy-
succinimidyl 2-[(triphenylmethylthio)methylcarbonylamino]etha-
noate (523 mg, 1.07 mmol) and DIPEA (0.19 mL, 1.07 mmol)
were added to a solution of 5 (120 mg, 1.07 mmol) in acetonitrile
(25 mL) and the mixture was refluxed overnight. After cooling to
room temperature and removal of the solvent, compound 8 was
recovered as a white powder by column chromatography on silica
gel (CH2Cl2–MeOH (95 : 5)). Yield: 251 mg (0.52 mmol, 49%).
Found: C, 65.8; H, 5.2; N, 11.0. C28H28N4O2S·0.5CH2Cl2
[ReO{pz-(CH2)2NH-Gly-CH2S}] (12). To a solution of com-
pound 8 (116 mg, 0.24 mmol) and ReOCl3(PPh3)2 (200 mg,
0.24 mmol) in dry methanol (20 mL) was added NEt3 (84 lL,
0.60 mmol), and the mixture was refluxed overnight. After
cooling to room temperature, the solvent was evaporated and the
crude product was purified by silica gel column chromatography
(CH2Cl2 : MeOH (95 : 5)), giving 12 as an orange solid. Yield:
159 mg (0.24 mmol, 98%).
1
requires C, 64.9; H, 5.5; N, 10.6%. IR: mmax/cm−1 1559 (CO). H
NMR (300 MHz, CDCl3): dH 3.14 (2H, s, CH2); 3.50 (2H, d,
J = 5.4 Hz, –COCH2N); 3.63 (2H, q, J = 5.4 Hz, CH2); 4.20
(2H, t, J = 5.1 Hz, CH2); 5.29 (1H, s, H(4)-pz); 6.46 (1 + 1H,
br, NH); 7.18–7.48 (15 + 2H, m, Ph-Trit + H(3/5)-pz). 13C NMR
(75.373 MHz, CDCl3): dC 35.5 (CH2); 39.6 (CH2); 43.4 (CH2); 50.6
(CH2); 105.7 (C(4)-pz), 127.04 (CH-Trit), 128.17 (CH-Trit), 129.39
(CH-Trit), 139.465 (C(3/5)-pz), 143.8 (C-Trit, C(3/5)-pz), 168.46
(CO), 168.65 (CO). Rf (s´ılica gel, CH2Cl2–MeOH (95 : 5)) = 0.30.
Found C, 25.2; H, 3.0; N, 12.3. C9H11N4O3SRe requires C, 24.5;
H, 2.5; N, 12.7. IR: mmax/cm−1 984 (Re O), 1628 (CO). H NMR
1
=
(300 MHz, dmso-d6): dH 3.17 (1H, t, J = 12.3 Hz, CH2); 3.83 (1H,
d, J = 17.1 Hz, CH2); 4.06 (1H, d, J = 17.1 Hz, CH2), 4.30 (1H, d,
J = 18 Hz, CH2); 4.60 (2H + 1H, m, CH2); 5.02 (1H, d, J = 18 Hz,
CH2); 6.86 (1H, t, H(4)-pz); 8.57 (1H + 1H, m, H(3/5)). 13C NMR
(75.373 MHz, dmso-d6): 38.0 (CH2); 46.8 (CH2); 53.7 (CH2); 55.90
(CH2); 109.65 (C(4)-pz); 138.40 (C(3/5)-pz), 148.32 (C(3/5)-pz);
188.65 (CO), 189.93 (CO). Rf (s´ılica gel, CH2Cl2–MeOH (95 : 5)) =
0.45. Rf (silica gel/MeOH) = 0.70. HPLC: tR = 14.00 min.
[ReO{3,5-Me2pz-(CH2)2NH-Gly-CH2S}] (13). Compound 13
is a carmine solid which was synthesized and purified in the
same way as that described above for 12, starting from 200 mg
(0.24 mmol) of ReOCl3(PPh3)2 and 123 mg (0.24 mmol) of ligand
11. Yield: 111 mg (0.24 mmol, 97%).
Found C, 27.8; H, 3.8; N, 11.7. C11H15N4O3SRe requires C, 28.1;
H, 3.2; N, 11.9. IR: mmax/cm−1 971 (Re O); 1636 (CO). H NMR
(300 MHz, CDCl3): dH 2.49 (3H, s, CH3-pz); 2.92 (3H, s, CH3-pz);
3.55 (1H + 1H, m, CH2); 3.91 (1H, d, J = 18 Hz, CH2); 4.04 (1H,
d, J = 18 Hz, CH2) 4.42 (1H, d, J = 18 Hz CH2); 4.49 (1H, m,
3,5-Me2pz(CH2)2NH-Gly-CH2STrit (9). Compound 9 is a
white solid which has been synthesized and purified as described
above for 8, starting from 121 mg (0.86 mmol) of 6. Yield: 274 mg
(0.53 mmol, 64%).
Found C, 62.5; H, 6.1; N, 9.5. C30H32N4O2S.CH2Cl2 requires
1
C, 62.3; H, 5.7; N, 9.4%. IR: mmax/cm−1 1675 (CO). H NMR
(300 MHz, CDCl3): dH 2.15 (3H, s, CH3-pz); 2.16 (3H, s, CH3-pz);
3.13 (2H, s, CH2); 3.50 (2H, d, J = 5.4 Hz, CH2); 3.58 (2H, q, J =
5.4 Hz CH2); 4.00 (2H, t, J = 5.4 Hz, CH2); 5.75 (1H, s, H(4)-pz);
6.51 (1H, br, NH); 6.55 (1H, br, NH); 7.17–7.21 (10H, m, Ph-Trit),
7.24–7.41 (5H, m, Ph-Trit). 13C NMR (75.373 MHz, CDCl3): 10.7
(pz-CH3); 13.5 (pz-CH3); 35.5 (CH2); 39.3 (CH2); 43.3 (CH2); 46.8
(CH2); 105.2 (H(4)-pz); 127.0 (CH-Trit), 128.14 (CH-Trit), 129.4
1
=
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CH); 4.66 (1H, m, CH2); 5.44 (1H, d, J = 18 Hz CH2); 6.40 (1H, s,
H(4)-pz). 13C NMR (75.373 MHz, CDCl3): dC 11.9 (CH3-pz), 16.9
(CH3-pz), 39.2 (CH2); 42.7 (CH2); 50.6 (CH2), 55.9 (CH2), 110.5
(C(4)-pz), 146.6 (C(3/5)-pz), 158.6 (C(3/5)-pz), 190.3 (CO), 191.2
(CO). Rf (s´ılica gel, CH2Cl2–MeOH (95 : 5)) = 0.50.
15 lL of SnCl2 in 0.1 N HCl (4 lg lL−1) and 800 lL of Na99mTcO4
(2–6 mCi) were successively added to the ligand solution. The final
preparation was incubated at room temperature for 30 min and
analysed by TLC and HPLC.
TLC (silica gel/MeOH): Rf = 0.7; HPLC: tR = 14.63 min.
[ReO{4-(EtOOC)CH2-3,5-Me2pz-(CH2)2NH-Gly-CH2S}] (14).
To a solution of [NBu4][ReOCl4] (27 mg, 0.045 mmol) in dry
methanol (5 mL) was added compound 10 (27 mg, 0.045 mmol)
dissolved in the minimum volume of methanol, followed by
addition of neat NEt3 (26 lL, 0,18 mmol) and the resulting mixture
was stirred for 2 h at room temperature. After this time, the solvent
was evaporated and the crude product purified by silica gel column
chromatography (ethyl acetate : methanol (90 : 10)), giving 14 as
a carmine solid. Yield: 20 mg (0.036 mmol, 80%).
Octanol–water partition coefficient. The log Po/w value of 12a
was determined by the multiple back extraction method under
physiological conditions (n-octanol/0.1 M PBS, pH 7.4).44 log
Po/w (12a) = + 0.73 0.03.
Glutathione challenge. In nitrogen purged vials, 200 lL of
freshly prepared solutions of reduced glutathione, 10 mM and
20 mM in PBS (pH = 7.4), were added to 200 lL aliquots of
◦
complex 12a. The samples were incubated at 37 C and aliquots
Found C, 32.1; H, 4.2; N, 7.2. C15H21N4O5SRe requires C, 32.4;
were analyzed by HPLC at 15 min, 1 h and 2 h time intervals.
H, 3.8; N, 7.2. IR: mmax/cm−1 980 (Re O); 1624 (C O); 1734
=
=
1
=
(C O). H NMR (300 MHz, CDCl3): dH 1.28 (3H, t, J = 6.9 Hz,
CH3), 2.47 (3H, s, CH3-pz), 2.89 (3H, s, CH3-pz), 3.54 (2H +
H + H, m, CH2), 4.14 (2H + 2H, m, CH2S, pzCH2COO), 4.34
(1H, d, J = 18.3 Hz, CH2), 4.50 (1H, m, CH2); 4.73 (1H, m,
CH2); 5.43 (1H, d, J = 18.3 Hz, CH2). 13C NMR (75.373 MHz,
CDCl3): 10.6 (CH3-pz), 14.2 (CH3-pz), 15.2 (CH2), 29.7 (CH2),
39.1 (CH2), 42.5 (CH2), 50.8 (CH2), 55.9 (CH2), 61.6 (CH2), 114.6
(C(4)-pz), 145.6 (C(3/5)-pz), 156.9 (C(3/5)-pz), 169.8 (CO), 190.4
(CO), 191.2 (CO). Rf (s´ılica gel, CH2Cl2–MeOH (95 : 5)) = 0.40.
Acknowledgements
Carolina Moura, Rute F. Vitor and Leonor Maria thank the
Foundation for Science and Technology (FCT) for BI, PhD
and Post-doctoral research grants, respectively. The authors
would like to acknowledge the FCT for financial support
(POCI/QUI/59588/2004).
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Synthesis and in vitro evaluation of [99mTcO{pz-(CH2)2NH-Gly-
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