A pyrazolylamine-phosphonate monoester chelator for the fac-[M(CO) 3]+ core (M = Re, 99mTc): Synthesis, coordination properties and biological assessment

Article abstract of DOI:10.1002/jlcr.1415

Aiming to develop new strategies for the labeling of hydroxyl-containing biomolecules with the organometallic core fac-[^99mTc(CO) ₃]⁺, we have prepared a new model bifunctional chelator, L4 (ethyl hydrogen (2-{[2-(3,5-dimethyl-1H-pyrazol-l-yl)ethyl]amino}ethyl) phosphonate), combining a pyrazolyl-amine chelating group and a monophosphonate ethyl ester function (-P(O)OHOEt). The phosphonate group allows metal stabilization, and, simultaneously, can be considered as a potential attachment site for a biomolecule. Reaction of L4 with the precursor [ ^99mTc(H₂O)₃(CO)₃]⁺ gave the model radiocomplex [^99mTc(CO)₃(k³-L4)] (6a). This radiocomplex was identified by comparing its chromatographic profile with that of the corresponding Re analog (6) under the same conditions, also prepared and fully characterized by the usual analytical techniques. Radiocomplex 6a is moderately lipophilic (log P_o/w = 1.07), presenting high stability in vitro without any measurable decomposition or ligand exchange, even in the presence of strong competing chelators such as histidine and cysteine (37°C, 24h). Biodistribution studies of the complex in CD-1 mice indicated a rapid blood clearance, and a rapid clearance from main organs, occurring primarily through the hepatobiliary pathway. Complex 6a presents also a high robustness in vivo, demonstrated by its resistance to metabolic degradation in blood, and intact excretion into the urine, after RP-HPLC analysis of blood and urine samples. Copyright

Full text of DOI:10.1002/jlcr.1415

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 1176–1184.
Published online 2 August 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1415
JLCR
A pyrazolylamine-phosphonate monoester chelator for the
fac-[M(CO)3]þ core (M ¼ Re, 99mTc): Synthesis, coordination
properties and biological assessment
´
˜
ELISA PALMA, BRUNO L. OLIVEIRA, FLAVIO FIGUEIRA, JOAO D. G. CORREIA, PAULA D. RAPOSINHO
and ISABEL SANTOS*
´
´
Departamento de Quımica, ITN, Estrada Nacional 10, 2686-953 Sacavem Codex, Portugal
Received 24 April 2007; Revised 17 May 2007; Accepted 1 June 2007
Abstract: Aiming to develop new strategies for the labeling of hydroxyl-containing biomolecules with the
organometallic core fac-½99mTcðCOÞ3ꢀþ, we have prepared a new model bifunctional chelator, L4 (ethyl hydrogen
(2-{[2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl]amino}ethyl)phosphonate), combining a pyrazolyl-amine chelating group
and a monophosphonate ethyl ester function (–P(O)OHOEt). The phosphonate group allows metal stabilization, and,
simultaneously, can be considered as a potential attachment site for a biomolecule. Reaction of L4 with the
precursor ½99mTcðH2OÞ3ðCOÞ3ꢀþ gave the model radiocomplex [99mTc(CO)3(k3-L4)] (6a). This radiocomplex was
identified by comparing its chromatographic profile with that of the corresponding Re analog (6) under the same
conditions, also prepared and fully characterized by the usual analytical techniques. Radiocomplex 6a is moderately
lipophilic (log Po=w ¼ 1:07), presenting high stability in vitro without any measurable decomposition or ligand
exchange, even in the presence of strong competing chelators such as histidine and cysteine (378C, 24 h).
Biodistribution studies of the complex in CD-1 mice indicated a rapid blood clearance, and a rapid clearance from
main organs, occurring primarily through the hepatobiliary pathway. Complex 6a presents also a high robustness
in vivo, demonstrated by its resistance to metabolic degradation in blood, and intact excretion into the urine, after
RP-HPLC analysis of blood and urine samples. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: Tc-99m; rhenium; phosphonate; tricarbonyl; pyrazolyl-amine; phosphazolidine
Introduction
properties.3 Promising BFCA includes tridentate
ligands, which comprise aliphatic or aromatic amines,
carboxylate groups, phosphines, thiols, thioethers and
others, giving rise to chelators with different charges
and donor-atom sets.1–5 Therefore, depending on the
combination of donating groups, cationic, neutral or
anionic complexes with different physicochemical
properties and pharmacokinetic profiles are formed.6
Moreover, the stability of the radioactive complexes
in vivo is of paramount importance, as dissociation of
the radiometal through transchelation processes or
reoxidation can lead to poor image quality, unneces-
sary radiation exposure to the patient and faulty image
interpretation. This issue gains utmost importance
when dealing with radionuclides for therapy.4 Thus,
The development of new organometallic radioactive
complexes based on the fac-½99mTcðCOÞ3ꢀþ moiety for
probing complex biochemical processes (e.g. receptor
binding and antigen expression), which proceed var-
ious diseases such as cancer, is a subject of intense
research.1–4 One of the most important key issues is
the availability of adequate bifunctional chelating
agents (BFCAs) with coordinating units for stabilization
of the radioactive metal core and, simultaneously,
pendant amino or carboxylic acid groups for the
linkage of important targeting molecules (e.g. peptides,
central nervous system receptor ligands, enzyme
substrates, etc.), while retaining their biological
the choice of the most adequate chelator for
a
particular targeting molecule must take into considera-
tion not only the physicochemical and biological
properties of the radioactive complexes, but also their
stability in vivo. Regarding phosphorus-containing
ligands for biomedical applications, Katti et al. have
*Correspondence to: Isabel Santos, Departamento de Qu´ımica, ITN,
Estrada Nacional 10, 2686-953 Sacave´m Codex, Portugal.
E-mail: isantos@itn.pt
Contract/grant sponsor: Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT);
contract/grant number: POCI/SAU-FCF/58855/2004
Contract/grant sponsor: Mallinkrodt-Tyco Inc.
Copyright # 2007 John Wiley & Sons, Ltd.

PYRAZOLYLAMINE-PHOSPHONATE MONOESTER CHELATOR 1177
developed tri- and tetradentate chelators combining
nitrogen or sulfur as donor atoms, and highly stable
tertiary phosphines, namely hydroxymethyl phos-
phines, which allowed the labeling of tumor-seeking
peptides with 99mTc for potential use in nuclear
medicine.7–10 Most recently, a new BFCA containing a
picolylamine-methylphosphonic acid ester group for
the labeling of hydroxyl-containing biomolecules with
the fac-½99mTcðCOÞ3ꢀþ core has been developed.11 Aim-
ing to develop new site-specific radiopharmaceuticals
based on the organometallic approach, we have intro-
duced in the past few years a new family of model
functionalize alcohol-containing biomolecules, forming
a stable phosphonic acid ester bond (Figure 2).
Herein, we describe the synthesis and characteriza-
tion of a new model chelator with a pyrazolyl-amine
framework and a monophosphonate ethyl ester group
(–P(O)OHOEt, L4) as well as the attempts undertaken
to prepare a chelator with a hydroxymethyl phosphine
motif (–P(CH2OH)2). The preparation, characterization
and biological evaluation of the organometallic com-
plexes fac-[M(CO)3(k3-L4)] (M ¼ Re, 6; 99mTc, 6a) are
also reported.
ligands for the stabilization of the fac-½MðCOÞ3ꢀþ
Results and discussion
99m
moiety (R ¼
Tc, Re), which contains a common
pyrazolyl-amine chelating unit (Figure 1), and an
aromatic (L1) or aliphatic (L2) amine, or a carboxylic
acid group (L3).12–15
Synthesis and characterization of diethyl (2-{[2-(3,5-
dimethyl-1H-pyrazol-1-yl)ethyl]amino}ethyl)phosph-
onate (3)
The model complexes fac-½99mTcðCOÞ3ðk3-LÞꢀþ=0
(L ¼ L1, L2 and L3), obtained by reaction of
the pyrazolyl-containing ligands L1–L3 with fac-
The synthetic pathway for the preparation of the
precursor compound
(Scheme 1).
3 involves two main steps
½
99mTcðCOÞ3ðH2OÞ3ꢀþ, display different physicochemical
and biological properties, and are remarkably stable
in vitro and in vivo, being adequate for the labeling of
different types of biologically active molecules.16,17 To
enable fine tuning of the physicochemical and biologi-
cal properties of the 99mTc model complexes and to
broaden the range of biomolecules that could be
labeled with pyrazolyl-containing ligands, we tried to
In the first general step, the pthalimide anion is
reacted with 1-(2-bromoethyl)-3,5-dimethyl-1H-pyra-
zole (1) to give the intermediate N-substituted
pthalimide, which upon treatment with hydrazine
monohydrate, followed by acid hydrolysis, gives the
corresponding primary amine 2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethanamine (2) in 82% yield. Selective
monoalkylation of the primary amine in 2 with diethyl
(2-bromo-ethyl)phosphonate under appropriate reaction
conditions gave (2-{[2-(3,5-dimethyl-1H-pyrazol-1-yl)
ethyl]amino}ethyl)phosphonate (3) in moderate yield
(32%) after purification by column chromatography.
develop new ligands combining
a pyrazolyl-amine
chelating unit and a phosphonic acid or an hydro-
xymethyl phosphine group. The main advantage of
the phosphonic acids in BFCA is their potential ability
to coordinate to the metal center and, simultaneously,
O
H
N
H
N
H
N
N
N
N
N
N
OH
N
N
N
L1
L2
L3
Figure 1 Ligands containing a pyrazolyl-amine chelating unit for the stabilization of the fac-½MðCOÞ3ꢀþ core (R ¼
Tc, Re).
99m
H
N
O
P
OH
OH
N
N
HO-Biomolecule
Coupling reagent
+1
O-Biomolecule
O
Tc
CO
OC
P
N
N
O
H
N
NH
Tc
CO
O
O
Biomolecule
N
P
OH
OC
CO
N
CO
Figure 2 Labeling of alcohol-containing biomolecules with 99mTc-tricarbonyl.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1176–1184
DOI: 10.1002.jlcr

1178 E. PALMA ET AL.
O
P
H
N
NH2
Br
OEt
OEt
N
N
N
N
N
)
iii
i )
ii)
N
1
2
3
Scheme 1 Synthesis of compound 3. (i) Potassium phtalimide/CH3CN, reflux, 72 h. (ii) Hydrazine monohydrate/MeOH, reflux,
3 h, 37% HCl. (iii) Diethyl (2-bromoethyl)phosphonate/CH3CN, K2CO3, reflux, 15 days.
Compound 3 has been characterized by 1H and 31P
NMR. The 1H-NMR spectrum (CDCl3) presents the
typical sharp singlet peaks for the H(4) proton (d 5.75)
and the methyl groups of the pyrazolyl ring (d 2.21 and
d 2.18), as well as resonances for the methylenic
protons and the diethylphosphonate group. A single
peak (d 30.5) for the phosphorus nucleus present in
the molecule is found in the 31P-NMR spectrum in the
same solvent.
H
N
O
OEt
OEt
N
P
N
3
ii)
i )
H
N
O
P
H
N
OEt
OH
N
N
PH2
N
N
4
L4
Synthesis and characterization of {1-[2-(3,5-dime-
thyl-1H-pyrazol-1-yl)ethyl]-1,3-azaphospholidin-3-yl}-
methanol (5) and ethyl hydrogen (2-{[2-(3,5-dimet-
hyl-1H-pyrazol-1-yl)ethyl]amino}ethyl)-phosphonate
(L4)
iii)
d
b
a
P
OH
N
N
f
c
e
N
5
The preparation of a chelator combining a pyrazolyl-
amine framework and a stable hydroxymethyl phos-
phine from precursor 3 has been attempted as outlined
in Scheme 2.
Scheme 2 Synthesis of compounds L4 and 5. (i) LiA1H4,
diethyl ether. (ii) 1M NaOH solution in EtOH, reflux, 3 h. (iii)
HCHO, EtOH, 30 min, room temperature (Identification
system for NMR assignments is displayed for 5).
Reduction of the phosphonate group in 3 with a 1 M
diethyl ether solution of lithium aluminum hydride
gave the intermediate compound 4, which contains
crucial for peak assignment in the NMR spectra, and
for full characterization of the compound.
a
phosphine hydride. Similar to what has been
previously described by Katti et al. for this type of
compounds, 4 presents a high degree of oxidative
stability upon contact with atmospheric oxygen and
water, and lack of reactivity toward solvents and
different type of chemical functionalities.7–10 The for-
mylation of the P–H bonds, carried out in degassed
ethanol with 37% aqueous formaldehyde gave, instead
of the hydroxymethyl phosphine groups, the rare
Partial alkaline hydrolysis of the diethyl phosphonate
ester group in 3 using a standard procedure (1 M NaOH
ethanolic solution, reflux) gave L4, after purification by
Sep-pak, as a white solid (Scheme 2), soluble in water
and alcohols. The chelator L4 has been thoroughly
characterized by the usual analytical techniques
in chemistry. The most characteristic peaks in the
1H-NMR spectrum (CD3OD) for this molecule appear at
d 5.88 and d 2.27/d 2.17, assigned to the H(4) proton
and the methyl groups of the pyrazolyl ring, respec-
tively. As expected, the chemical shifts, multiplicity and
relative intensity of the peaks at d 3.97–3.87 (m, CH2,
2H) and d 1.25 (t, CH3, 3H), assigned to one ethyl
group, are in full agreement with the presence of a
monophosphonate ethyl ester function (–P(O)OHOEt)
in the molecule. The 13C spectrum shows 11 peaks,
which correspond to all the carbon nuclei of the ligand.
A single peak in the 31P-NMR spectrum is found at
d 20.8. The purity of the isolated compound (>95%)
has been ascertained by analytical reversed phase
high-performance liquid chromatography (RP-HPLC).
heterocyclic phosphazolidine compound
5
(yield:
92%). This intramolecular cyclization is not surprising
since it is known that the condensation of primary
phosphines with formaldehyde in the presence of
secondary amines leads, in a Mannich-type reaction,
to tertiary phosphines containing dialkylaminoethyl
groups or to phosphazolidine derivatives.18,19 Com-
pound 5 was obtained as an air-stable, pale-yellow
clear oil, and has been thoroughly characterized by
FTICR-MS (Fourier transform ion cyclotron resonance
mass spectrometry) and 1H/13C/31P-NMR spectro-
scopy (see Experimental part). The use of 2D-NMR
1
experiments (1H-1H COSY and H-13C HSQC) has been
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1176–1184
DOI: 10.1002.jlcr

PYRAZOLYLAMINE-PHOSPHONATE MONOESTER CHELATOR 1179
diastereotopic character of some of the methylenic
protons of the ligand backbone upon coordination to
the metallic center (d 4.54, CH2-1, d 4.04, CH2-10; d
3.04, CH2-2; d 2.74, CH2-20). This behavior, already
observed in other tricarbonyl complexes, is typical
Synthesis and characterization of the Re tricarbonyl
complex fac-[Re(CO)3(k3-L4)] (6)
The bifunctional chelator L4 reacts with the organo-
metallic precursor fac-[Re(CO)3(H2O)3]þ in refluxing
water or methanol yielding the neutral complex 6
(ca. 46%) after purification by semi-preparative RP-
HPLC (Scheme 3). This complex has been synthesized
as a ‘non-radioactive’ analog of the corresponding
99mTc complex (6a).
for asymmetric ligands containing
a
pyrazolyl-
amine backbone, being a strong evidence of ligand
coordination.12–14,20 The 13C-NMR spectra showed
splitting due to the C–P coupling for some of the aliphatic
carbon atoms. Coupling to the C-4 (1JPC-4 ¼ 7:5 Hz) and
2
to the C-5 (isomer a: JPC-5 ¼ 7:4 Hz isomer b:
Complex 6 is an air-stable white solid, which has
been characterized by electrospray ionization mass
spectrometry (ESI-MS; m/z: 546 ½M þ Hꢀþ, 568
½M þ Naꢀþ) and NMR spectroscopy (1H/13C/31P-NMR,
1H-1H COSY, and 1H-13C HSQC). The NMR studies and
the RP-HPLC chromatograms clearly indicated the
formation of two isomers (isomer a: ret. time 18.6 min,
isomer b: ret. time 18.9 min) in the ratio 4:6, most likely
formed due to the different positions assumed by the
ester group on the coordinating phosphonate unit.
The coordination mode (k3-N,N,O) of L4 toward the
‘Re(CO)3’ moiety, and the presence of the rare
P–O–ReI(CO)3 coordination, has been established
based on the NMR spectroscopic data of 6. This
coordination mode compares well with the one found
in the organometallic compound fac-[Re(CO)3(k3-L)]
(L ¼ picolylamine–mehylphosphonic acid ester), which
has been characterized both in solution by NMR, and in
the solid state by X-ray crystallography.11 In this
complex, the crystal structure revealed the presence
of a delocalized electron pair shared by the endstanding
P–O bond and the P–O bond involved in the coordina-
tion to the metallic center.
In the 1H-NMR spectra (CD3OD) of enriched mixtures
in each isomer, obtained by RP-HPLC purification, the
most striking features are the different chemical shifts
observed for the H(4)pz signal (isomer a: d 6.12, isomer
b: d 6.09), consistent with the involvement of the azole
ring in the coordination to the metal, and for the
protons of the phosphonate ethyl ester (isomer a: d
3.79, OCH2CH3; d 1.15, OCH2CH3. isomer b: d 4.04,
OCH2CH3; d 1.37, OCH2CH3) in both isomers. Another
important feature of the spectrum is related with the
2JPC-5 ¼ 12:1 Hz) is strong for both isomers. The 31P-
NMR spectrum in CD3OD shows singlets at d 25.6 and d
23.5 assigned to isomers a and b, respectively. These
downfield shifted signals (D ¼ 4:8 ppm, isomer a;
D ¼ 2:7 ppm, isomer b) relative to free L4 ligand, is an
evidence for P–O–ReI(CO)3 coordination.
Synthesis and characterization of fac-[99mTc(CO)3
(k3-L4)] (6a)
The complex fac-[99mTc(CO)3(k3-L4)] (6a) was obtained
in ca. 75% yield at 1008C, using ligand concentrations
in the 10ꢁ5–10ꢁ4 M range. The only radiochemical
impurities identified by RP-HPLC were [99mTcO4]ꢁ and
½
99mTcðCOÞ3ꢀþ. The chemical identity of complex 6a
after RP-HPLC purification was confirmed by compar-
ing its HPLC profile (retention time: 18.8 min) with that
of the corresponding rhenium complex 6 (Figure 3).
Surprisingly, no evidence for the formation of isomers
of the radioactive complex 6a was found.
Complex 6a is moderately lipophilic (log Po=w ¼ þ1:07),
and displays high in vitro stability in the presence of
excess of cysteine, histidine or PBS pH 7.4, since HPLC
5
6
OCH2CH3
4
1
3
2
OH2
Re
+1
P
N
NH
Re
O
H2O
OC
OH2
CO
L4
O
+
N
OC
CO
CO
CO
6
Scheme 3 Preparation of the rhenium tricarbonyl complex
fac-[Re(CO)3(k3-L4)] (6). (Numbering system for NMR assign-
ments is displayed).
Figure
3 RP-HPLC analytical chromatograms of 6 (UV
detection, ret. time ¼ 18:6 min, isomer a; 18.9 min, isomer b)
and 6a (g detection, ret. time ¼ 18:8 min).
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1176–1184
DOI: 10.1002.jlcr

1180 E. PALMA ET AL.
analysis revealed that no trans-chelation or reoxidation
to pertechnetate occurred, even after 24 h at 378C. The
stability profile is similar to that of other complexes
anchored on pyrazolyl-diamine chelators previously
reported.12–17,20
(1 h p.i.) were analyzed by radiometric RP-HPLC. The
murine serum, isolated from blood does not show any
traces of pertechnetate, and more than 98% of the
radioactivity could be assigned to the complex. Analy-
sis of urine collected at the same time demonstrated
again high in vivo stability for the complex, because no
metabolites could be detected (Figure 4). The resistance
to metabolic degradation in blood and the excretion of
the 99mTc tricarbonyl complex 6a intact into the urine
demonstrate its high in vivo robustness.
Biodistribution of 6a. The organ distribution of purified
6a in female CD-1 mice as a function of time is
summarized in Table 1.
Complex 6a shows a rapid blood clearance as only
0.87 ꢂ 0.20% ID of the administered dose remained in
whole blood at 1 h p.i., and a rapid clearance from
main, non-excretion, organs. Comparatively, 6a
cleared more rapidly than the analogous complex fac-
Conclusion
We have synthesized and characterized a new chelator
containing a pyrazolyl-amine backbone and a phos-
phonate monoester (L4) for stabilization of the fac-½M
ðCOÞ3ꢀþ core (M ¼ Re, 99mTc). Reaction of this novel
bifunctional chelating ligand with the corresponding
organometallic precursor afforded the model radio-
complex fac-[99mTc(CO)3(k3-L4)] (6a) showing that L4
can be potentially useful for the labeling of biomolecules
containing an alcohol function. Complex 6a has been
identified by comparing with the corresponding Re
surrogate (6), which has also been synthesized and fully
characterized. Biodistribution studies of radiocomplex
[
99mTc(CO)3(k3-L3)] stabilized by the pyrazolyl-contain-
ing ligand L3, which presents similar physico-chemical
properties (moderate lipophilicity and neutral
charge).15 As expected from the lipophilic character of
both complexes (log Po/w: 1.07 and 1.10 for 6a and fac-
[
99mTc(CO)3(k3-L3)], respectively) they were mainly
excreted by the hepatobiliary pathway. Consequently,
the overall excretion rate was relatively slow, in
accordance with what is normally found for lipophilic
radiocomplexes. However, the radiocomplex 6a was
excreted faster (54.5% after 4 h p.i.) than fac-
6a in female CD-1 mice indicated
a rapid blood
[
99mTc(CO)3(k3-L3)] (29.7% after 4 h p.i.). Indeed, after
clearance, and a relatively rapid clearance from main
organs, being excreted mainly through the hepatobiliary
pathway. The radiocomplex is stable both in vitro and
in vivo, without any measurable decomposition or
reoxidation. It is still necessary to optimize the labeling
yields of the chelator in order to expand the use of the
promising bifunctional chelator L4 to the labeling of a
large variety of hydroxyl-containing biomolecules.
4 h, the hepatic uptake observed for 6a was relatively
low (4.88 ꢂ 0.38% ID per organ), most of the radio-
activity being already in the intestine (37.83 ꢂ 13.28%
ID per organ). There was no significant uptake
or retention of radioactivity in the stomach
(0.10 ꢂ 0.03%/g, 4 h, p.i.), demonstrating that 6a does
not undergo in vivo reoxidation to [99mTcO4]ꢁ.
To further verify the in vivo stability of 6a, samples of
murine serum and urine collected at sacrifice time
Materials and methods
Table 1 Biodistribution data of complex 6a in female CD-1
mice (n ¼ 5) at 1 and 4 h postinjection (p.i.) (% IDgꢁ1 ꢂ stan-
dard deviation)
All chemicals and solvents were of reagent grade and
were used without purification unless stated otherwise.
18.8 min
Tissue
6a
1 h
4 h
Blood
Liver
Intestine
Spleen
Heart
0.63 ꢂ 0.15
7.74 ꢂ 1.76
22.99 ꢂ 2.34
0.19 ꢂ 0.03
0.30 ꢂ 0.06
0.46 ꢂ 0.10
2.77 ꢂ 0.45
0.22 ꢂ 0.02
0.15 ꢂ 0.02
0.48 ꢂ 0.16
0.43 ꢂ 0.15
0.20 ꢂ 0.02
3.82 ꢂ 0.41
15.54 ꢂ 4.90
0.07 ꢂ 0.01
0.12 ꢂ 0.04
0.18 ꢂ 0.02
1.12 ꢂ 0.24
0.07 ꢂ 0.01
0.06 ꢂ 0.01
0.10 ꢂ 0.03
0.15 ꢂ 0.03
Lung
Kidney
Muscle
Bone
Stomach
Pancreas
0
5
10
15
20
25
30
Figure 4 RP-HPLC radioactive trace (g-detection) for an urine
sample collected at 1 h p.i.
Total excretion (%)
29.7 ꢂ 2.9
54.5 ꢂ 14.1
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

PYRAZOLYLAMINE-PHOSPHONATE MONOESTER CHELATOR 1181
Diethyl (2-bromoethyl)phosphonate was obtained in
high yield (>85%) using the classical Michaelis–Arbu-
zov reaction, i.e. by refluxing dry triethyl phosphite
in a 30-fold excess of dry dibromoethane overnight,
followed by vacuum distillation.21 The precursors
1-(2-bromoethyl)-3,5-dimethyl-1H-pyrazole (1) and
[Re(CO)3(H2O)3]Br were prepared according to pub-
lished methods.22,23 Na[99mTcO4] was eluted from a
99Mo/99mTc generator, using 0.9% saline. The radio-
active precursor fac-½99mTcðCOÞ3ðH2OÞ3ꢀþ was prepared
using a IsoLink1 kit (Malinckrodt, Inc.). 1H, 11C NMR
and 31P spectra were recorded at room temperature on
a Varian Unity 300 MHz spectrometer. 1H and 11C
chemical shifts were referenced with the residual
solvent resonances relative to tetramethylsilane, and
the 31P-NMR chemical shifts with external 85% H3PO4
solution. NMR spectra were run in CDCl3, CD3OD and
D2O. HPLC analyses were performed on a Perkin Elmer
LC pump 200 coupled to a Shimadzu SPD 10AV UV/
Vis detector and to a Berthold-LB 507A radiometric
detector, using an analytic Macherey–Nagel C18 re-
versed-phase column (Nucleosil 100-10, 250 ꢃ 4 mm)
and a gradient of aqueous 0.1% CF3COOH/MeOH with
a flow rate of 1 mL/min.
Synthesis of 2-(3,5-dimethyl-1H-pyrazol-1-yl)ethana-
mine (2): Hydrazine monohydrate (9.8 mL, 0.20 mol)
was added to a suspension of 2-[2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl]-1H-isoindole-1,3(2H)-dione (5.21 g,
0.019 mol) in MeOH (150 mL). After refluxing for 3 h,
the reaction mixture was allowed to cool to room
temperature, and 37% HCl (43.5 mL) was added
dropwise. The white solid formed was removed by
filtration and the pH of the resulting solution was
adjusted to pH 9 with 3 M NaOH. The aqueous phase
was extracted with CHCl3 (3 ꢃ 100 mL). The organic
phases were collected, dried over MgSO4, filtered, and
concentrated to dryness, giving an yellow viscous
oil which crystallized on standing. Yield: 2.21 g, 82%.
1H-NMR (CDCl3): d 5.75 (s, H(4)pz, 1H); 3.97 (t br., CH2,
2H); 3.06 (t br., CH2, 2H); 2.20 (s, CH3, 3H), 2.17
(s, CH3, 3H), 1.63 (s br., NH2, 2H).
Synthesis of diethyl (2-{[2-(3,5-dimethyl-1H-pyrazol-
1-yl)ethyl]amino}ethyl)phosphonate (3)
Diethyl (2-bromoethyl)phosphonate (1.092 g, 4.46 mmol)
and 2-(3,5-dimethyl-1H-pyrazol-1-yl)ethanamine (0.414 g,
2.97 mmol) were dissolved in CH3CN (25 mL) and an
excess of K2CO3, (0.821 g, 5.94 mmol), KI (catalytic
amount) were added. After 15 days under reflux, the
suspended solids were removed by filtration, and water
(ca. 30 mL) was added to the solution. The reaction
mixture was extracted with CH2Cl2 (3 ꢃ 75 mL). The
organic phases were collected, dried over MgSO4,
filtered and concentrated to dryness. The residue was
purified by column chromatography (MeOH (5–50%)/
CH2Cl2). The product was obtained as a pale-yellow oil.
Yield: 0.62 g, 69%. 1H-NMR (CDCl3): d 5.75 (s, H(4)pz,
1H); 4.09–4.02 (m, CH2, 6H); 3.00 (t, CH2, 2H); 2.93–
2.84 (m, CH2, 2H) 2.21 (s, CH3, 3H), 2.18 (s, CH3, 3H),
1.99–1.88 (m, CH2, 2H); 1.28 (t, CH3, 6H). 31P NMR
(CDCl3): d 30.5.
Gradient: t ¼ 0–3 min: 0% MeOH; 3–3.1 min: 0 ! 25%
MeOH; 3.1–9 min: 25% MeOH; 9–9.1 min: 25 ! 34%
MeOH; 9.1–19 min: 34 ! 100% MeOH; 19–21 min:
100 ! 0% MeOH; 21–30 min: 0% MeOH. Purification
of the inactive compounds was achieved on a semi-
preparative Macherey–Nagel C18 reversed-phase
column (Nucleosil 100-7, 250 ꢃ 8 mm) using a gradient
of aqueous 0.1% CF3COOH/CH3CN as eluent and
a flow rate of 2 mL/min. Gradient: t ¼ 0–5 min: 20%
CH3CN; 5–30 min: 20 ! 100% CH3CN; 30-34 min:
100% CH3CN; 34–35 min: 100 ! 20% CH3CN; 35–40 min:
20% CH3CN.
Synthesis of 2-(3,5-dimethyl-1H-pyrazol-1-yl)ethana-
mine (2)
Synthesis of ethyl hydrogen (2-{[2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl]amino}ethyl)phosphonate (L4)
Synthesis of 2-[2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl]-
1H-isoindole-1,3(2H)-dione: To a solution of 1-(2-bromo-
ethyl)-3,5-dimethyl-1H-pyrazole (1, 9.56 g, 0.047 mol)
in dry CH3CN (200 mL) potassium phtalimide (26.14 g,
0.141 mol) was added. After 72 h under reflux and a
nitrogen atmosphere, the solution was decanted and
filtered. The clear solution obtained was evaporated to
dryness and the residue recrystallized from MeOH,
giving the compound as a white solid. Yield: 8.68 g,
69%. 1H-NMR (CDCl3): d 7.86–7.76 (m, CH aromatic,
2H); 7.74–7.67 (m, CH aromatic, 2H), 5.75 (s, H(4)pz,
1H); 4.26 (t, CH2, 2H); 4.01 (t, CH2, 2H); 2.18 (s, CH3,
3H), 2.06 (s, CH3, 3H).
The monoethyl ester was obtained by alkaline hydro-
lysis of 3 (0.105 g, 0.35 mmol) in a 1 M NaOH/EtOH
solution (2 mL). After 3 h of reflux, the solution was
cooled and the reaction concentrated to half of the
initial volume. The solution was then neutralized to pH
7 with 1 M HCl and the solvents removed under
reduced pressure until a white solid was obtained.
The solid was extracted with three portions of EtOH.
After evaporation of the solvent, L4 was obtained as a
white solid. The compound was further purified by Sep-
pak: the cartridge was washed with water (2 mL) and
the product was eluted with 2:1 methanol:water (2 mL).
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1176–1184
DOI: 10.1002.jlcr

1182 E. PALMA ET AL.
Evaporation of the eluate afforded the product as a
white solid. Yield: 0.08 g, 83%. 1H-NMR (CD3OD): d
5.88 (s, H(4)pz, 1H); 4.26 (t, CH2, 2H); 3.97–3.87 (m,
CH2, 2H); 3.44 (t, CH2, 2H); 3.26–3.17 (m, CH2, 2H);
2.27 (s, CH3, 3H), 2.17 (s, CH3, 3H), 1.96–1.85 (m, CH2,
2H); 1.25 (t, CH3, 3H). 13C NMR (CD3OD) d 149.9 (C(3/
5)pz), 141.7 (C(3/5)pz), 106.7 (C(4)pz), 61.1 (d,
J ¼ 5:7 Hz, POCH2CH3), 45.1 (d, J ¼ 26:2 Hz, H2CP),
25.8 (CH2), 25.2 (CH2), 24.0 (CH2), 17.1 (d, J ¼ 6:6 Hz,
H(4)pz, 1H); 3.98 (t, CH2-a, 2H); 3.71–3.53 (m, CH2-e,
2H); 2.96–2.86 (m, CH2-b, 1H); 2.80 (t, CH2-c+CH2-f,
2H+1H); 2.60–2.46 (m, CH2-f0, 1H); 2.36–2.26 (m, CH2-
b0, 1H), 2.08 (s, CH3, 3H), 1.98 (s, CH3, 3H), 1.92–1.76
(m, CH2-d, 1H); 1.68–1.59 (m, CH2-d0, 1H); 13C-NMR
(D2O) d 148.2 (C(3/5)pz), 140.8 (C(3/5)pz), 104.5
(C(4)pz), 61.0 (d, 1JPC ¼ 19 Hz, C-e), 54.0 (d,
2
3JPC ¼ 2 Hz, C-c) 53.4 (d, JPC ¼ 4 Hz, C-b), 51.4 (d,
1
1JPC ¼ 13 Hz, C-f), 45.6 (C-a), 19.1 (d, JPC ¼ 10 Hz, C-
POCH2CH3), 13.4 (CH3), 10.7 (CH3). 31P NMR (CD3OD):
d), 11.4 (CH3), 9.5 (CH3). 31P-NMR (D2O): d ꢁ28.5.
FTICR-MS(þ) m/z: 242 ½M þ Hꢀþ. See identification
system for NMR assignment in Scheme 2.
%
d 20.8. Anal. Calc. for C11H21N3O3PNa: C, 44.44; H,
7.12; N, 14.13. Found: C, 45.11; H, 6.57; N, 14.07.
ESI-MS (þ): m/z 276 ½M þ Hꢀþ, 298 ½M þ Naꢀþ. Reten-
tion time (RP-HPLC): 9.7 min.
Synthesis of [Re(CO)3(k3-L4)] (6)
Synthesis of 2-(3,5-dimethyl-1H-pyrazol-1-yl)-N-(2-
phosphinoethyl)ethanamine (4)
To a solution of [Re(CO)3(H2O)3]Br (0.047 g, 0.12 mmol)
in MeOH (10 mL) L4 (0.032 g, 0.012 mmol) was added,
and the resulting mixture was refluxed overnight. After
evaporation of the solvent, the residue obtained was
dried under vacuum and analyzed by RP-HPLC. The
crude product revealed a mixture of unreacted Re
precursor (retention time: 6.3 min), free L4 (retention
time: 9.7 min) and complex 6 as two isomers (retention
time: isomer a – 18.6 min, isomer b – 18.9 min). The
isomers were separated and isolated by RP-HPLC. Yield
(both isomers): 30 mg, 46%.
The compound has been prepared according to a
slightly modified reported method9: To a stirred cold
(08C) solution of LiAlH4 (2.2 mL, 1 M solution in diethyl
ether), a suspension of the diethyl phosphonate 3
(0.328 g, 1.08 mmol) in dry diethyl ether (24 mL) in a
nitrogen atmosphere was added. The reaction was
allowed to react for 4 h at room temperature; the
mixture was cooled to 08C and the excess of LiAlH4
was eliminated by slow addition of cold aqueous brine
solution (20 mL), followed by the addition of aqueous
KOH solution (10%, 20 mL). The organic layer was
separated, the aqueous layer was extracted with ether
(1 ꢃ 20 mL) and CH2Cl2 (2 ꢃ 25 mL), and the organic
phases were collected and washed with brine (80 mL).
After drying over anhydrous MgSO4, the solvents were
evaporated in the rotary evaporator under reduced
pressure and the colorless oil obtained was vacuum
dried. The product is stable for some weeks if stored at
ꢁ208C. Yield: 0.14 g, 65%. 1H-NMR (CDCl3): d 5.75 (s,
H(4)pz, 1H); 4.03 (t, CH2, 2H); 2.98 (t, CH2, 2H); 2.90 (t,
PH, 1H); 2.74 (q, CH2, 2H); 2.25 (t, PH, 1H); 2.20 (s,
CH3, 3H), 2.17 (s, CH3, 3H), 1.66–1.58 (m, CH2, 2H).
31P NMR (CDCl3): d ꢁ147.1.
Isomer a: 1H-NMR (CD3OD): d 6.22 (s br., 1H, NH),
6.12 (s, 1H, H(4)pz), 4.54 (dd, J ¼ 4:5 Hz, J ¼ 15:4 Hz,
1H, CH2-1), 4.04 (m, 1H, CH2-10), 3.79 (p, J ¼ 7:1 Hz,
2H, OCH2-5), 3.47 (m br, 2H, CH2-4), 3.04 (m, 1H, CH2-
2), 2.74 (m, 1H, CH2-20), 2.52 (s, 3H, CH3), 2.34 (s, 3H,
CH3), 1.79 (m br., 2H, CH2-3), 1.15 (t, J ¼ 7:4 Hz, 3H,
CH3-6). 13C-RMN (CD3OD): d 197.0–194.5 (3 ꢃ CꢄO),
154.5 (C(3/5)pz), 143.9 (C(3/5)pz), 108.4 (C(4)Pz), 62.1
(d, 2JPC ¼ 7:4 Hz, C-5), 52.1 (d, 1JPC ¼ 7:5 Hz, C-4), 50.6
(C-2), ꢅ49.0 (C-1, overlapped with CD3OD as con-
2
firmed by HSQC), 23.8 (d, JPC ¼ 22:3 Hz, C-3), 16.9
(C-6); 15.2 (CH3pz); 11.4 (CH3pz). 31P-NMR (CD3OD):
d 25.6.
Isomer b: 1H-NMR (CD3OD): d 6.22 (s br., 1H, NH),
6.09 (s, 1H, H(4)pz), 4.54 (dd, J ¼ 4:5 Hz, J ¼ 15:4 Hz,
1H, CH2-1,), 4.04 (m, 3H, CH2 ꢁ 10 þ OCH2 ꢁ 5), 3.59
(m br., 2H, CH2-4), 3.04 (m, 1H, CH2-2), 2.74 (m, 1H,
CH2-20), 2.47 (s, 3H, CH3), 2.33 (s, 3H, CH3), 1.85 (m,
2H, CH2-3), 1.37 (t, J ¼ 7:4 Hz, 3H, CH3-6). 13C-RMN
(CD3OD): d 197.0–194.5 (3 ꢃ CꢄO), 154.7 (C(3/5)pz),
Synthesis of {1-[2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl]-
1,3-azaphospholidin-3-yl}-methanol (5)
An aqueous solution of formaldehyde (0.040 ml of a
37% solution) was placed in oxygen-free ethanol
(2.5 mL) and purged with nitrogen gas for some
minutes. This solution was added dropwise with
stirring at room temperature to compound 4 (0.048 g,
0.24 mmol). The resulting solution was allowed to react
for 30 min at room temperature. Removal of the solvent
in vacuo afforded compound 5 as a colorless, viscous
oil. Yield: 0.052 g, 92%. 1H-NMR (D2O): d 5.76 (s,
2
143.9 (C(3/5)pz), 108.4 (C(4)Pz), 60.7 (d, JPC ¼ 12:1
1
Hz C-5), 52.6 (d, JPC ¼ 7:5 Hz, C-4), 50.4 (C-2),
ꢅ49.0 (C-1, overlapped with CD3OD as confirmed
by HSQC), 21.9 (d, 2JPC ¼ 22:4 Hz, C-3), 16.8
(C-6), 15.4 (CH3pz), 11.4 (CH3pz). 31P-NMR (CD3OD):
d 23.5. In both isomers, there is occasional overlapping
of some resonances associated with the two isomers, as
confirmed by two-dimensional NMR techniques (1H-1H
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1176–1184
DOI: 10.1002.jlcr

PYRAZOLYLAMINE-PHOSPHONATE MONOESTER CHELATOR 1183
COSY and 1H-13C HSQC). See numbering system for
NMR assignments in Scheme 3. ESI-MS (þ): m/z 546
½M þ Hꢀþ, 568 ½M þ Naꢀþ.
purified complex 6a via the tail vein, and were
maintained on normal diet ad libitum. Mice were
sacrificed by cervical dislocation at 1 and 4 h p.i. The
injected radioactive dose and the radioactivity remain-
ing in the animal after sacrifice were measured in a
dose calibrator (Aloka, Curiemeter IGC-3, Tokyo,
Japan). The difference between the radioactivity in
the injected and sacrificed animal was assumed to be
due to total excretion from whole animal body. Blood
samples were taken by cardiac puncture at sacrifice.
Tissue samples of the main organs were removed,
weighed and counted in a gamma counter (Berthold).
Biodistribution results were expressed as a percen-
tage of the injected dose per gram tissue (%ID/g). For
blood, bone and muscle, total activity was calculated
assuming that these organs constitute 6, 10 and 40%
of the total weight, respectively. The remaining
activity in the carcass was also measured in a dose
calibrator.
Synthesis of the 99mTc-complex 6a
A volume of 900 mL of the organometallic precursor fac-
½
99mTcðOH2Þ3ðCOÞ3ꢀþ in saline pH 7.4 and 100 mL of an
aqueous solution of L4 (8 ꢃ 10ꢁ4 M) were placed in a
5-mL glass vial under nitrogen. The vial was then
heated to 1008C for 60 min, cooled on an ice bath and
the final solution analyzed by RP-HPLC. Retention
time: 18.8 min. Complex 6a was purified for the
biodistribution studies using
a
semi-preparative
Knauer C18 reversed-phase column (Hypersil 120
ODS 10 mm, 250 ꢃ 8 mm) and a gradient of aqueous
0.1% CF3COOH/MeOH with a flow rate of 2 mL/min.
Gradient: t ¼ 0–3 min: 0% MeOH; 3–3.1 min: 0 ! 25%
MeOH; 3.1–9 min: 25% MeOH; 9–9.1 min: 25 ! 34%
MeOH; 9.1–19 min: 34 ! 100% MeOH; 19–21 min:
100 ! 0% MeOH; 21–30 min: 0% MeOH. The peak
corresponding to the complex was collected in a Falcon
flask containing 200 mL of saline. The solvent was
removed under a stream of nitrogen until a final volume
of 200 mL was reached. The solution was diluted to the
desired radioactivity with PBS pH 7.4, and the product
analyzed again by analytical RP-HPLC. This compound
has also been used for the in vitro stability studies.
Assessment of the in vivo stability of 6a by RP-HPLC
analysis
The in vivo stability of 6a was assessed by urine and
murine serum RP-HPLC analysis, under the same
chromatographic conditions used for 6a. The samples
were obtained 1 h p.i. from the biodistribution experi-
ment. The urine collected at sacrifice time was filtered
through a Millex GV filter (0.22 mm) before RP-HPLC
analysis. Blood collected from mice was immediately
centrifuged at 3000 rpm for 15 min at 48C, and the
serum was separated. The serum was treated with
ethanol in a 2:1 (v/v) ratio to precipitate the proteins.
After centrifugation at 3000 rpm for 15 min at 48C, the
supernatant was filtered through a Millex GV filter
(0.22 mm) and analyzed by RP-HPLC.
Octanol–water partition coefficient
The log Po/w values of complexes 6a were determined by
the multiple back extraction method under physiologi-
cal conditions (n-octanol/0.1 M PBS, pH 7.4).24
Cysteine and histidine challenge
In total, 100 ml of purified 6a was added to 900 mL of
10ꢁ3 M cysteine or histidine solutions in PBS at pH 7.4.
The samples were incubated at 378C and analyzed by
RP-HPLC after 24 h.
Acknowledgements
This work has been partially supported by the
Fundac¸a˜o para a Cieˆncia e Tecnologia (FCT) through
the project POCI/SAU-FCF/58855/2004. We thank
Mallinkrodt-Tyco Inc. for the financial support.
Bruno L. Oliveira e Elisa Paula would like to thank
FCT for BI grants. We wish to acknowledge the Mass
Spectrometry Laboratory for performing the ESI-MS
determinations at the Instituto de Tecnologia Qu´ımica
e Biolo´gica, Universidade Nova de Lisboa, Oeiras,
Portugal.
Biodistribution studies
All animal studies were conducted in accordance with
the highest standards of care, as outlined in the
National and European Law. The animals were housed
in a temperature- and humidity-controlled room with a
12 h light/12 h dark schedule. The in vivo behavior of
complexes 6a was evaluated in groups of five female
CD-1 mice (randomly bred, Charles River) weighing
approximately 20–25 g each. Animals were injected
intravenously with 100 mL (25–30 mCi or 50–60 mCi for
1 and 4 h biodistribution timepoints, respectively) of
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