A 99mTc(CO)3-labeled benzylguanidine with persistent heart uptake

Article abstract of DOI:10.1002/jlcr.3188

We describe the synthesis and biological evaluation of the cationic ^99mTc-tricarbonyl complex fac-[^99mTc(CO) ₃(κ³-L1)]⁺ (Tc1) anchored by a pyrazole-diamine-methylbenzylguanidine-based ligand (L1), as potentially useful for myocardial imaging. The rhenium complex fac-[Re(CO)₃(κ ³-L1)]⁺ (Re1) was prepared and characterized as a 'cold' surrogate of the radioactive complex. Cell uptake studies in a neuroblastoma cell line suggest that Tc1 uptake mechanism is related to the norepinephrine transporter (NET). Tissue distribution studies in CD1 mice showed that Tc1 presents high initial heart uptake and a slow washout from the heart (7.8 ± 1.3% injected dose per gram (ID/g), 30-min post-injection (p.i.); 6.3 ± 1.3% ID/g, 60-min p.i.), with heart to blood ratios of 11.8 and 9.0 at 30- and 60-min p.i., respectively. The uptake mechanism of Tc1 appears to be similar to that of metaiodobenzylguanidine (MIBG), as it can be reduced by coinjection with nonradioactive MIBG. The biodistribution profile of Tc2, where the benzylguanidine pharmacophore is absent, corroborates the fact that Tc1 does not accumulate in the heart by a simple diffusion mechanism but rather by a NET-mediated mechanism. The results confirm those obtained in the cell assays. Despite the persistent heart uptake found for Tc1, the high hepatic and renal uptake remains to be improved. Copyright

Full text of DOI:10.1002/jlcr.3188

Research Article
Received 11 December 2013,
Revised 07 January 2014,
Accepted 08 January 2014
Published online 17 February 2014 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3188
99m
A Tc(CO)3-labeled benzylguanidine with
persistent heart uptake‡
†
*
Bruno L. Oliveira, Maurício Morais, Lurdes Gano, Isabel Santos,
*
and João D. G. Correia
We describe the synthesis and biological evaluation of the cationic 99mTc–tricarbonyl complex fac-[99mTc(CO)3(κ3-L1)]+ (Tc1)
anchored by a pyrazole-diamine-methylbenzylguanidine-based ligand (L1), as potentially useful for myocardial imaging. The
rhenium complex fac-[Re(CO)3(κ3-L1)]+ (Re1) was prepared and characterized as a ‘cold’ surrogate of the radioactive
complex. Cell uptake studies in a neuroblastoma cell line suggest that Tc1 uptake mechanism is related to the
norepinephrine transporter (NET). Tissue distribution studies in CD1 mice showed that Tc1 presents high initial heart uptake
and a slow washout from the heart (7.8 1.3% injected dose per gram (ID/g), 30-min post-injection (p.i.); 6.3 1.3% ID/g,
60-min p.i.), with heart to blood ratios of 11.8 and 9.0 at 30- and 60-min p.i., respectively. The uptake mechanism of Tc1
appears to be similar to that of metaiodobenzylguanidine (MIBG), as it can be reduced by coinjection with nonradioactive
MIBG. The biodistribution profile of Tc2, where the benzylguanidine pharmacophore is absent, corroborates the fact that
Tc1 does not accumulate in the heart by a simple diffusion mechanism but rather by a NET-mediated mechanism. The results
confirm those obtained in the cell assays. Despite the persistent heart uptake found for Tc1, the high hepatic and renal
uptake remains to be improved.
Keywords: metaiodobenzylguanidine; myocardial imaging; norepinephrine transporter; radiopharmaceuticals; rhenium; technetium-99m
transporter (NET), and its uptake matches the density and
Introduction
integrity of sympathetic neurons.22–25 Radioiodinated MIBG has
The most used radiotracers in the clinical setting for myocardial
been also widely used for the diagnosis of neuroendocrine
imaging are the cationic 99mTc-radiopharmaceuticals, 99mTc-sestamibi,
and 99mTc-tetrofosmin. However, their pharmacokinetic profile is
far from ideal. Indeed, they show high liver uptake, which can
interfere in the analysis of cardiac imaging, particularly of the
inferior left ventricular wall.1–6 In the past years, intense research
efforts have been focused on the development of novel cationic
99mTc-based probes with improved heart to background ratios.
The most promising results have been obtained with the mixed-
ligand 99mTc(V)-nitrido complexes stabilized by bisphosphino-
amine (PNP)-type ligands such as 99mTc-N-DBODC5, 99mTc-N-15C5,
and 99mTc-N-MPO. Promising results were also attained with the
99mTc(CO)3-complex and 99mTc-15C5-PNP.7–11 Recently, we have
investigated the coordination chemistry of pyrazole-based ligands with
the ‘99mTc(CO)3’ core, and we were able to identify a new class of
organometallic complexes based on tri-methoxy-tris-pyrazolylmethane
(TMEOP) potentially useful for myocardial imaging.12–15 Among these
77/
tumors.26,27 Radiohalogenated analogs of MIBG such as
76Bromo-labeled and 18Fluoro-labeled guanidines, as well as
11C-labeled guanidines, have been also explored as potential
myocardial imaging agents.26,28–32 With regard to the targeting
of the NET with 99mTc(V)-complexes for single photon emission
computed tomography imaging of sympathetic neurons, Kung
et al. have introduced a set of 99mTc(V)-labeled MIBG derivatives,
containing chelating groups of the N2S2 type, which compare
well with radioiodinated MIBG in terms of heart uptake.33
The numerous advantages of the ‘M(CO)3’ core for
radiopharmaceutical applications prompted us to develop a
99mTc(I)-MIBG derivative potentially useful as a heart imaging
agent.34–38 In this context, we designed a novel bifunctional
chelator (L1), which combines a tridentate pyrazolyl-diamine
chelating backbone for metal stabilization and a pendant
complexes, fac-[99mTc(CO)3{κ3-HC[3,4,5-(CH3OCH2)3pz]3}]+
(
99mTc-tri-
Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade
de Lisboa, Estrada Nacional 10 (ao km 139, 7)2695-066 Bobadela LRS, Portugal
methoxy-tris-pyrazolylmethane (TMEOP)) showed a high heart uptake
and a biodistribution profile suitable for myocardial imaging.13,14
Their heart uptake mechanism is similar to that of other reported
monocationic 99mTc-based cardiac agents and is associated with
accumulation in the mitochondria of myocytes.15–19
Another family of radiotracers successfully applied to
myocardial imaging is represented by radioiodinated
metaiodobenzylguanidine (MIBG), an analog of norepinephrine,
which is used for scintigraphic imaging studies of cardiac
sympathetic innervations.20,21 MIBG is actively transported into
presynaptic sympathetic nerve terminals by the norepinephrine
*Correspondence to: João D. G. Correia and Bruno L. Oliveira, Centro de Ciências
e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa,
Estrada Nacional 10 (ao km 139, 7), 2695-066 Bobadela LRS, Portugal.
E-mail: jgalamba@itn.pt; oliveira@nmr.mgh.harvard.edu
† Current address: A. A. Martinos Center for Biomedical Imaging, Massachusetts
General Hospital and Harvard Medical School, 149 13th Street, Suite 2301,
Charlestown, MA 02129 (USA).
‡Additional supporting information may be found in the online version of this
article at the publisher's website.
J. Label Compd. Radiopharm 2014, 57 358–364
Copyright © 2014 John Wiley & Sons, Ltd.

B. L. Oliveira et al.
benzylguanidine moiety for probing the NET. The conjugate Synthetic procedures
L1 allowed the synthesis of the MIBG derivative fac-[99mTc(CO)3
(κ3-L1)]+ (Tc1), whose biological properties were assessed in a
neuroblastoma cell line as well as in CD1 mice.
Preparation of tert-butyl 2-((4-cyanobenzyl)(2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl)amino)-ethylcarbamate (2)
A solution of 1 (0.600 g, 2.125 mmol), 3-(bromomethyl)benzonitrile
(0.833 g, 4.250 mmol), and K2CO3 (0.587 g, 4.250 mmol) in acetonitrile
(ACN) (35 mL) was refluxed for 24 h. After the reaction, the solution was
filtered and the solvent evaporated to give an oily residue, which was
purified by silica gel column chromatography using a gradient of EtOAc
(0 → 100%) in hexane. The fractions containing 2 were collected, and the
compound was obtained as colorless oil after evaporation of the solvent.
Yield: 55.6% (0.470 g, 1.183 mmol). 1H-NMR (300 MHz, CDCl3): δH (ppm)
7.45 (2H, m, CHm/k), 7.29 (2H, m, CHn/j), 6.00 (1H, br m, NHBoc), 5.82
(1H, s, CHb), 4.00 (2H, t, J = 7.8 Hz, CH2d), 3.55 (2H, s, CHh2), 3.20 (2H, m,
CHe2), 2.70 (2H, t, J = 7.6 Hz, CHf2), 2.61 (2H, t, J = 7.6 Hz, CHg2), 2.28 (3H,
s, CHa3/c), 1.98 (3H, s, CH3a/c), and 1.44 (9H, s, C(CH3)3). 13C-NMR
(75.5 MHz, CDCl3): δc (ppm) 156.6 (C=O, Boc), 147.5 (Cc), 141.2 (Ca),
138.7 (Ci), 133.0 (Ck/m), 132.0 (Ck/m), 130.8 (Cj/n), 129.1 (Cj/n), 119.12
(Cl), 112.6 (CC≡N), 105.7 (Cb), 79.1 (C(CH3)3, 58.0 (Ch), 53.4 (Ce), 53.3 (Cf),
46.7 (Cd), 38.6 (Cg), 28.7 (C(CH3)3, 13.6 (CH3a/c), and 10.9 (CHa3/c). IR (KBr,
cmÀ1): 2229vw ν(C≡N), 1711 m, and 1175 m.
Experimental
General procedures and materials
All chemicals and solvents were of reagent grade and were used without
purification unless stated otherwise. The Boc-protected precursors tert-
butyl (2-{[2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl]amino}ethyl)carbamate
(1) and [Re(CO)3(H2O)3]Br were prepared according to published
methods.39,40 All other chemicals not specified previously were
purchased from Aldrich. 1H and 13C NMR spectra were recorded at room
temperature on a Varian Unity 300-MHz spectrometer. 1H and 13C
chemical shifts were referenced with the residual solvent resonances
relatively to tetramethylsilane. The spectra were assigned with the help
of 2D experiments (1H–1H correlation spectroscopy, and 1H–13C
heteronuclear single quantum coherence). Assignments of the 1H and
13C NMR resonances are given in accordance with the identification
system shown in Scheme 1. Infrared (IR) spectra were recorded as KBr
pellets on
a Bruker Tensor 27 spectrometer. Compounds were
Preparation of tert-butyl 2-((4-(aminomethyl)benzyl)(2-(3,5-dimethyl-
1H-pyrazol-1-yl)ethyl)-amino)ethylcarbamate (3)
characterized by electrospray ionization mass spectrometry (ESI-MS)
using a quadrupole ion trap mass spectrometer (QITMS) instrument.
Aliquots of ~5 mg of pure compounds ≥95% ascertained by reversed
phase HPLC (RP-HPLC) were lyophilized in eppendorf tubes and used
for radioactive labeling and in vitro studies. Na[99mTcO4] was eluted from
NiCl2 · 6H2O (0.023 g, 0.096 mmol) was added to a solution of 2 (0.382 g,
0.961 mmol) in MeOH (10 mL) cooled with an ice bath to give a green
solution. NaBH4 (0.290 g, 7.688 mmol) was added in portions with stirring,
and the resulting purple mixture was stirred overnight at 20°C. The
solvent was evaporated and the residue partitioned between EtOAc
and a saturated NaHCO3 solution. The organic phase was dried over
MgSO4. Removal of organic solvent gave a colorless residue. Compound
3 was used without any further purification. Yield: 79.5% (0.307 g,
0.764 mmol). 1H-NMR (300 MHz, CDCl3): δH (ppm) 7.17–6.98 (4H, m, CHj/
k/m/n), 5.75 (1H, s, CHb), 5.50 (2H, br m, NH2), 3.98 (2H, s, CHo2), 3.78 (2H,
m, CHd2), 3.56 (2H, s, CH2h), 3.14 (2H, m, CH2e), 2.76 (2H, t, J = 7.7 Hz, CHf2),
2.56 (2H, t, J = 7.7 Hz, CH2g), 2.23 (3H, s, CH3a/c), 2.02 (3H, s, CHa3/c), and
1.42 (9H, s, C(CH3)3). 13C-NMR (75.5 MHz, CDCl3): δc (ppm) 156.5 (C = O,
Boc), 147.5 (Cc), 139.5 (Ca), 139.2 (Cl), 128.8 (Cj/n), 128.7 (Cj/n), 127.3 (Ck/m),
127.4 (Ck/m), 125.9 (Ci), 105.2 (Cb), 79.1 (C(CH3)3, overlapped with CDCl3),
58.9 (Ce), 53.4 (Cf), 53.3 (Cd), 47.0 (Co/h), 45.0 (Co/h), 38.6 (Cg), 28.7 (C(CH3)3),
13.7 (CH3a/c), and 11.1 (CH3a/c). IR (KBr, cmÀ1): 1713 m and 1176 m.
a
99Mo/99mTc generator using 0.9% saline. HPLC analyses were
performed on a Perkin Elmer LC pump 200 coupled to a Shimadzu SPD
10AV ultraviolet–visible (UV/Vis) and to a Berthold LB 509 radiometric
detector, using an analytic Macherey–Nagel C18 reversed-phase column
(Nucleosil 100-5, 250 × 3 mm) with a flow rate of 0.5mL/min. Purification
of the inactive compounds was achieved on
a semipreparative
Macherey–Nagel C18 reversed-phase column (Nucleosil 100-7,
250 × 8 mm) with a flow rate of 2.0 mL/min. UV detection: 220 or 254nm.
Eluents: A = aqueous 0.1% CF3COOH and B = MeOH. Gradient of method 1:
t = 0–3 min, 0% B; 3–3.1 min, 0 →25% B; 3.1–9 min, 25% B; 9–9.1min,
25 →34% B; 9.1–20 min, 34 → 100% B; 20–25min, 100% B; 25–25.1 min,
100 →0% B; and 25.1–30min, 0% B. Gradient of method 2: t = 0–5 min,
10% MeOH; 5–30min, 10 →100% MeOH; 30–34 min, 100% MeOH; 34–
35 min, 100 → 10% MeOH; and 35–40min, 10% MeOH.
Scheme 1. Synthesis of L1 and L2. (i) K2CO3, KI, ACN, reflux, 24 h, 55.6%; (ii) NaBH4, NiCl2, MeOH, r.t., overnight, 79.5%; (iii) 1H-pyrazole-1-carboximidamide, DIPEA, DMF,
overnight, 76.4%; (iv) TFA, CH2Cl2, r.t., 2 h, 38.8%; and (v) TFA, CH2Cl2, r.t., 2 h, 55.5% (identification system for NMR spectra assignment is displayed for L1 and L2).
J. Label Compd. Radiopharm 2014, 57 358–364
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

B. L. Oliveira et al.
Preparation of tert-butyl 2-((2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl) General procedure for the preparation of Re(I) complexes of
(4-(guanidinomethyl)-benzyl)amino)ethylcarbamate (4)
the type fac-[Re(CO)3(κ3-L)] (Re1, L = L1; Re2, L = L2).
A solution of 3 (0.065 g, 0.162 mmol), 1H-pyrazole-1-carboximidamide
(0.036 g, 0.243 mmol), and N,Nʹ-diisopropylethylamine (DIPEA) (0.031 g,
0.243 mmol) in dimethylformamide (DMF) was stirred for 48 h. After the
reaction, the DMF was evaporated to give an oily residue, which was
purified by silica gel column chromatography using a gradient of MeOH
(0 → 100%) in CHCl3. The fractions containing 4 were collected, and the
compound was obtained as pale yellow oil after evaporation of the
elution solvents. Yield: 76.4% (0.055 g, 0.123 mmol). 1H-NMR (300 MHz,
CD3OD): δH (ppm) 7.29–7.10 (4H, m, CHj/k/m/n), 5.80 (1H, s, CHb), 4.34
(2H, s, CHo2), 4.00 (2H, t, J = 7.7 Hz, CH2d), 3.33 (2H, s, CHh2), 3.07 (2H,
m, CHe2), 2.77 (2H, t, J = 7.6 Hz, CH2f ), 2.58 (2H, t, J = 7.6 Hz, CHg2), 2.15
(3H, s, CHa3/c), 2.13 (3H, s, CH3a/c), and 1.31 (9H, s, C(CH3)3). 13C-NMR
(75.5 MHz, CD3OD): δc (ppm) 157.5 (p), 157.1 (C=O, Boc), 154.0 (Cc),
144.0 (Ca), 140.1 (Cl), 129.3 (Cj/n), 128.7 (Cj/n), 128.3 (Ck/m), 127.6
(Ck/m), 125.9 (Ci), 104.8 (Cb), 78.8 (C(CH3)3), 58.5 (Ce), 53.6 (Cf), 53.3
(Cd), 46.6 (Co/h), 44.9 (Co/h), 38.2 (Cg), 27.6 (C(CH3)3), 11.7 (CHa3/c),
[Re(CO)3(H2O)3]Br was reacted with 1.2 equivalents of the compounds L1
and L2 in refluxing H2O for 18 h. After reacting, the solvent was removed
under vacuum, and the resulting residue was dissolved in water and
purified by semipreparative RP-HPLC (method 2).
Synthesis of fac-[Re(CO)3(κ3-L1)] (Re1 · 2TFA)
Starting from 0.045 g (0.078 mmol) of L1 · 2TFA, a colorless oil formulated
as Re1 was obtained. Yield: 76.2% (0.050 g, 0.059 mmol, calcd. for
C21H30N7O3Re · 2CF3COOÀ). 1H-NMR (300 MHz, CD3OD): δH (ppm)
7.42–7.27 (4H, m, CHj/k/m/n), 6.00 (1H, s, CHb), 5.23 (1H, m, NH), 4.48 (2H,
dd, J= 7.2Hz, CHh2), 4.33 (2H, s, CH2o), 4.13 (2H, m, CH2d), 4.13 (2H, m, CHd2′),
3.74 (1H, m, NH), 3.20 (1H, m, CHe2), 3.00 (2H, m, CH2g), 2.56 (1H, m, CHe2′),
2.45–2.33 (2H, m, CH2f,f′), 2.25 (3H, s, CH3a/c), and 2.21 (3H, s, CHa3/c). 13C-NMR
(75.5MHz, CD3OD): δc (ppm) 194.6 (C≡O), 194.0 (C≡O), 193.0 (C≡O), 157.1
(Cp), 154.1 (Cc), 144.4 (Ca), 137.0 (Cl), 132.6 (Cj/n), 132.2 (Cj/n), 131.2 (Ck/m),
129.3 (Ck/m), 128.0 (Ci), 105.0 (Cb), 69.2 (Cf), 61.1 (Ch), 51.3 (Ce), 46.1 (Cd),
44.4 (Cg), 41.8 (Co), 14.4 (CHa3/c), and 10.8 (CHa3/c). ESI-MS (+) (m/z): 614.2
[M]+, calcd. for C21H29N7O3Re= 614.2. IR (KBr, cmÀ1): 2028 and
1911 ν(C≡O). HPLC (Rt): 22.4 min (λ = 254nm, method 2).
1
and 9.9 (CHa3/c). IR (KBr, cmÀ ): 1700 s and 1169 m. Analytic HPLC
(Rt): 18.2 min (λ = 220 nm; method 1).
Preparation of 1-(4-(((2-aminoethyl)(2-(3,5-dimethyl-1H-pyrazol-1-yl)
ethyl)amino)methyl)-benzyl)guanidine (L1)
Synthesis of fac-[Re(CO)3(κ3-L2)] (Re2 · 2TFA)
Treatment of 4 (0.045 g, 0.101 mmol) with CH2Cl2/TFA (1 mL/2 mL) gave a
colorless oil corresponding to L1 after purification by semipreparative
Starting from 0.025 g (0.047 mmol) of L2 · 2TFA, a colorless oil formulated
as Re2 was obtained. Yield: 78.7% (0.030 g, 0.037 mmol, calcd. for
RP-HPLC. Yield: 37.6% (0.022 g, 0.038 mmol, calcd. for
C18H31N7 · 2
CF3COOÀ). 1H-NMR (300 MHz, CD3OD): δH (ppm) 7.24–7.03 (4H, m,
CHj/k/m/n), 5.86 (1H, s, CHb), 4.30 (2H, s, CH2o) 4.08 (2H, t, J = 7.7Hz, CHd2),
3.71 (2H, s, CHh2), 3.04 (2H, m, CHe2), 2.88 (2H, t, J = 7.7Hz, CHf2), 2.80 (2H, t,
J = 7.7 Hz, CH2g), 2.20 (3H, s, CH3a/c), and 2.00 (3H, s, CHa3/c). 13C-NMR
(75.5MHz, CD3OD): δc (ppm) 157.5 (Cp), 147.0 (Cc), 140.4 (Ca), 138.7 (Cl),
136.6 (Cj/n), 129.1 (Cj/n), 128.3 (Ck/m), 127.8 (Ck/m), 126.4 (Ci), 105.3 (Cb),
58.6 (Ce), 52.6 (Cf/g), 50.8 (Co/h), 45.7 (Cd), 44.8 (Co/h), 37.1 (Cf/g),
11.8 (CHa3/c), and 9.4 (CH3a/c). ESI-MS (+) (m/z): 344.2 [M + H]+, calcd.
for C18H29N7 = 343.2. Analytic HPLC (Rt): 15.2min (λ = 220 nm; method 1).
IR (KBr, cmÀ1): 3600 m, 1678 vs, 1205 m and 1141 w.
C
20H28N5O3Re · 2CF3COOÀ). 1H-NMR (300 MHz, CD3OD): δH (ppm)
7.46–7.20 (4H, m, CHj/k/m/n), 6.08 (1H, s, CHb), 5.20 (1H, m, NH), 4.46 (2H,
dd, J = 7.3 Hz, CHh2), 4.33 (1H, m, CH2d), 4.13 (2H, s, CHo2), 3.80 (1H, m, NH),
3.25–3.07 (4H, m, CHd2′, CH2e, NH), 2.56 (1H, m, CHe2′), 2.45–2.33 (2H, m,
CHg2), 2.33 (3H, s, CHa3/c), and 2.20 (3H, s, CHa3/c). 13C-NMR (75.5 MHz,
CD3OD): δc (ppm) 195.9 (C≡O), 194.6 (C≡O), 194.3 (C≡O), 156.4 (Cc),
144.4 (Ca), 133.6 (Cl), 133.3 (Cj/n), 133.2 (Cj/n), 133.0 (Ck/m), 132.6 (Ck/m),
132.5 (Ci), 105.0 (Cb), 68.6 (Cf), 60.8 (Ch), 50.5 (Ce), 45.9 (Cd), 43.0 (Cg),
41.8 (Co), 15.4 (CH3a/c), and 10.8 (CHa3/c). ESI-MS (+) (m/z): 572.2 [M]+, calcd.
for C20H27N5O3Re = 572.2. HPLC (Rt): 19.8 min (λ = 254 nm, method 2).
Preparation of N1-(4-(aminomethyl)benzyl)-N1-(2-(3,5-dimethyl-1H-
pyrazol-1-yl)ethyl)ethane-1,2-diamine (L2)
Determination of the partition coefficient (Log Po/w
)
Evaluated by the ‘shake flask’ method,41 Tc1 was added to a mixture of
octanol (1 mL) and 0.1 M PBS pH = 7.4 (1 mL), previously saturated in each
other by stirring the mixture. This mixture was vortexed and centrifuged
(3000 g, 10 min, room temperature) to allow phase separation. Aliquots of
both octanol and PBS were counted in a gamma counter. The partition
coefficient (Po/w) was calculated by dividing the counts in the octanol
Treatment of 3 (0.055 g, 0.137 mmol) with CH2Cl2/TFA (1 mL/2 mL) gave a
colorless oil corresponding to L2 after purification by semipreparative
RP-HPLC. Yield: 55.5% (0.040 g, 0.076 mmol, calcd. for
C17H29N5 · 2
CF3COOÀ). 1H-NMR (300 MHz, D2O): δH (ppm) 7.26–6.99 (4H, m, CHj/k/m/n),
5.82 (1H, s, CHb), 4.17 (2H, t, J = 7.7 Hz, CH2d), 4.12 (2H, s, CHh2), 4.05 (2H,
s, CH2o), 3.17 (6H, m, CH2e+f+g), 2.02 (3H, s, CH3a/c), and 2.00 (3H, s, CHa3/c).
13C-NMR (75.5 MHz, D2O): δc (ppm) 148.3 (Cc), 141.5 (Ca), 139.8 (Cl),
136.1 (Cj/n), 130.5 (Cj/n), 129.4 (Ck/m), 129.1 (Ck/m), 126.0 (Ci), 105.7 (Cb), 57.8
(Ce), 52.3 (Cf/g), 52.0 (Co/h), 46.24 (Cd), 46.20 (Co/h), 37.3 (Cf/g), 12.2 (CHa3/c),
and 9.8 (CHa3/c). ESI-MS (+) (m/z): 302.3 [M + H]+, calcd. for C17H27N5 = 301.2.
Analytic HPLC (Rt): 14.6min (λ = 220 nm; method 1). IR (KBr, cmÀ1): 3360 m,
1684 vs, 1640 sh, 1205 vs, and 1139 s.
phase by those in the buffer, and the results were expressed as Log Po/w
Tc1, Log Po/w = À0.763 0.021; Tc2, Log Po/w = À0.889 0.447.
.
Cell assays
SH-SY5Y human neuroblastoma cells were grown in Dulbecco’s modified
Eagle medium containing GlutaMAX I supplemented with 10% heat-
inactivated fetal bovine serum and 1% penicillin/streptomycin antibiotic
solution (all from Gibco, Invitrogen, UK). Cells were cultured in a
humidified atmosphere of 95% air and 5% CO2 at 37°C, with the medium
changed every 2 days. The cells were adherent in monolayers and, when
confluent, were harvested from the cell culture flasks with trypsin–EDTA
(Gibco, Invitrogen, UK) and seeded into 24-well plates [(2–4) × 105 cells/
per well in 500 μL of medium] and incubated for 24 h at 37°C. On the
General procedure for the preparation of Tc(I) complexes of
the type fac-[Tc(CO)3(κ3-L)] (Tc1, L = L1; Tc2, L = L2).
The radioactive precursor fac-[99mTc(CO)3(H2O)3]+ was prepared by
addition of Na[99mTcO4] to an IsoLink® kit (Mallinckrodt-Covidean, Inc.)
following a described procedure 38. In a nitrogen-purged glass vial, day of the experiment, the medium was discarded and replaced by fresh
900 μL of fac-[99mTc(CO)3(H2O)3]+ (1–2 mCi) in saline pH 7.4 was added medium that contained the radioactive compound. The cells were
to 100 μL of a 10À3 or 10À4 M aqueous solution of the compounds L1
or L2. The reaction mixture was then heated to 100°C for 30 min, cooled
exposed to Tc1 for 15, 30 min, 1, and 2 h at 37°C. Incubation was
terminated by washing the cells with ice-cold assay medium, washed
on an ice bath, and the final solution was analyzed by RP-HPLC. Retention twice with cold PBS, and subsequently, the cells were lysed by 10-min
times (method 2): 5.0 min for fac-[99mTc(CO)3(H2O)3]+, 23.1 min for Tc1,
and 20.6 min for Tc2.
incubation with 1 N NaOH at 37°C. The percent of cell-associated
radioactivity as a function of time was determined by counting in a
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B. L. Oliveira et al.
Berthold LB 2111 gamma counting system. Uptake studies were carried
out using at least four wells for each data point. Blocking studies were
performed by preincubating cells with DMI (50 μM) for 30 min.
Boc-deprotection of 4 and 3 under standard conditions afforded
L1 and L2, respectively. The latter ligand, where the guanidine
moiety is absent, was synthesized with the aim of preparing
the complex fac-[99mTc(CO)3(κ3-L2)]+, which will be used as a
negative control in the biodistribution studies. All compounds
were fully characterized by ESI-MS and 1H/13C-NMR and IR
spectroscopy.
The reduction of the C≡N group in 2 was confirmed by
1H-NMR and IR spectroscopy. Indeed, the absence of the
absorption band at 2229 cmÀ1 in the IR spectrum, assigned to
the stretching vibrations of the C≡N group, and the appearance
Biodistribution studies in mice
All animal experiments were performed in compliance with Portuguese
Law and European Directives (Portaria 1131/97, Decreto-Lei nº 129/92 de
6 de Julho e 197/96 de 16 de Outubro and 86/609/CEE) regarding ethics,
care, and protection of animals used for experimental and other scientific
proposes. The animals were housed in a temperature (22–23°C) and
humidity (45–65%) controlled room with a 12-h light/12-h dark schedule.
Biodistribution of the 99mTc complexes Tc1 and Tc2 was evaluated in of two new resonances at δ 5.50 (2H, br m) and 3.98 (2H, s) in the
1H-NMR spectrum (CDCl3), assigned to the additional amine and
groups of 3–4 CD1 female mice (Charles River outbred strain, from IFFA
CREDO, Barcelona, Spain). Mice with 22–29-g weight were intravenously
CH2 groups, respectively, indicated that the formation of 3.
1H-NMR spectra of L1 (CD3OD) and L2 (D2O) showed resonances
injected with 100 μL of each radiolabeled complex (in PBS) (60–90μCi) via
for the pyrazole-diamine backbone (L1: δ 5.86, 1Hb; δ 4.08, 2Hd; δ
the tail vein, and the animals were kept in normal diet ad libitum. Blocking
studies were carried out by coinjecting nonradioactive MIBG (1mg/kg)
3.04, 2He; δ 2.88, 2Hf; δ 2.80, 2Hg; δ 2.20, 3HCH3pz; δ 2.00, 3HCH3pz
together with Tc1. The effect of L-lysine on nonspecific kidney uptake of
and L2: δ 5.82, 1Hb; δ 4.17, 2Hd; 3.17, 6He+f+g; 2.02, 3HCH3pz
;
radiocomplex Tc1 was checked up by coinjection with L-Lys (15mg/50μl
saline) in order to block the cationic transporters on renal tubules. Animals
were sacrificed by cervical dislocation at 2, 30 and 60 min after injection.
The administered dose and the radioactivity in the sacrificed animals
δ 2.00, 3HCH3pz) and for the methyl-benzyl-guanidine (δ 7.24–7.03,
4Har; δ 4.30, 2Ho; δ 3.71, 2Hh) or methyl-benzyl-amine arms
(δ 7.26–6.99, 4Har; δ 4.12, 2Hh; δ 4.05, 2Ho). The 13C-NMR spectra
were measured with a dose calibrator (Curiemeter IGC-3, Aloka, Tokyo, of L1 and L2 display all the expected resonances, in particular,
Japan or Carpintec CRC-15W, Ramsey, USA). The difference in
radioactivity between the injected animal and the sacrificed animal
was assumed to be because of excretion. Tissues and organs of interest
were dissected, rinsed to remove excess blood, weighted, and their
radioactivity was measured using the dose calibrator or a γ-counter.
The uptake in the tissues or organs was calculated and expressed as
percent injected dose per gram of tissue or organ (% ID/g). For blood,
bone, and muscle, total activity was estimated assuming that they
represent 6, 10, and 40, of the total body weight, respectively. Blood
samples were taken by cardiac puncture at sacrifice. The blood was then
the presence of the carbon atom of the guanidine moiety in L1
(δ 157.5), which was not present in L2.
The complexes fac-[99mTc(CO)3(κ3-L1)]+ (Tc1) and fac-[99mTc
(CO)3(κ3-L2)]+ (Tc2) were synthesized by reaction of L1 and L2,
respectively, with the organometallic precursor fac-[99mTc(CO)3
(H2O)3]+ (Scheme 2), which was prepared using an IsoLink® kit
(Mallinckrodt, Covidien). The kit formulation, available for
research purposes, contains boranocarbonate ([H3BCO2]2À),
Na/K tartrate, sodium tetraborate decahydrate, and sodium
centrifuged, and the serum was separated and treated for HPLC analysis. carbonate. The boranocarbonate reduces the Tc(VII) and acts
Urine was also collected and pooled together at sacrifice time.
simultaneously as a CO source, through mechanisms not yet
fully understood.
Both complexes were obtained in high radiochemical yield
(>95%) and high radiochemical purity (>95%), and the chemical
identity was established by comparing their γ-traces (HPLC
radiochromatograms) with the UV/Vis traces of the respective
Re surrogates Re1 and Re2. For the sake of example, we present
the traces of the match pair Tc1/Re in Figure 1.
The complexes Re1 and Re2 were prepared by reaction of L1
and L2 with fac-[Re(CO)3(H2O)3]Br (Scheme 2), respectively. The
use of Re complexes to identify the molecular structure of the
99mTc congeners, by means of HPLC comparison, is a common
practice in radiopharmaceutical chemistry because of the
similar physicochemical properties of these group 7 elements.
In vivo stability
The in vivo stability of Tc1 was evaluated by RP-HPLC analysis of mice
urine and blood samples. The urine was collected at the sacrificed time,
centrifuged at 3000 g for 15 min and analyzed by RP-HPLC. The blood
was collected at the sacrificed time, centrifuged at 3000 g for 15 min at
4°C, and the serum was separated. One hundred microliter aliquots of
serum were sampled and treated with 200 μl of ethanol to precipitate
the proteins. Samples were then centrifuged at 3000 g for 15 min at 4°C.
The supernatant was analyzed by RP-HPLC.
Results and discussion
The synthetic pathway for the preparation of conjugate L1 is The complexes were fully characterized by the common
displayed in Scheme 1. Direct alkylation of precursor 1 with spectroscopic techniques, and the data collected were
3-(bromomethyl)benzonitrile, followed by reduction of the consistent with a κ3-N3 coordination mode for the pyrazolyl-
resulting intermediate 2 with NaBH4/NiCl2 gave 3, which upon diamine chelator, as found in other similar Re(I) complexes
reaction with 1H-pyrazole-1-carboximidamide afforded4(Scheme 1). already described by our group.42–45
Scheme 2. Preparation of fac-[99mTc(CO)3(κ3-L1)]+ (Tc1) and fac-[99mTc(CO)3(κ3-L2)]+ (Tc2).
J. Label Compd. Radiopharm 2014, 57 358–364
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B. L. Oliveira et al.
The complexes Tc1 and Tc2 display a moderate hydrophilic
nature as revealed by the partition coefficient (P) between 1-
octanol and phosphate buffer pH 7.0 expressed as Log Po/w
=
0.763 0.021 and Log Po/w = À0.889 0.447, respectively.
Aiming to assess the NET-recognizing ability of the
benzylguanidine-containing compounds L1 and metallated
congener Tc1, we have performed a cell uptake study in SH-
SY5Y cells. This cell line is a cloned subline of the neuroblastoma
cells SK-N-SH that express monoamine transporters and were
reported to take MIBG by an active mechanism.46–48 The results
from this study indicated a moderate Tc1 uptake by SH-SY5Y
cells that increased as a function of incubation time at 37°C
(Figure 2). To assess the specificity of Tc1 uptake, blocking
studies with desipramine (DMI, 50 μM), a selective NET inhibitor,
were carried out. A lower nonspecific uptake was found (~10–
20%) suggesting that the mechanism of the uptake may be
partially related with this transporter (Figure 2).
Figure 1. HPLC chromatograms of coinjected rhenium complex (Re1) (UV detection)
and 99mTc complex (Tc1) (γ detection).
Encouraged by the cell uptake results, we have evaluated the
biological behavior of Tc1 in CD1 mice to assess its relevance as
a potential radiotracer for myocardial imaging. The results of the
biodistribution studies performed at 2-, 30-, and 60-min
postinjection (p.i.) are presented in Table 1.
The complex showed a high heart uptake and a rapid
clearance from the blood stream and the lung. After the initial
high uptake (above 7.2% ID/g), a slight washout at the 1-h time
point was found, but the radioactivity level remained persistently
above 6% ID/g, resulting in a heart to blood ratio greater than
nine. The heart uptake (6.3 1.3% ID/g) is comparable to that
observed for 99mTc-sestamibi, a radiopharmaceutical in clinical
use for myocardial imaging, in the same animal model
(8.0 0.6% ID/g, 1-h p.i.).49 However, the uptake in the heart is
threefold lower than that of [125I]MIBG at 1-h p.i. in mice.50 It is
Figure 2. Uptake of Tc1 in SH-SY5Y neuroblastoma cells as a function of
incubation time (Tc1) and in the presence of desipramine (Tc1 + DMI). All results
are presented as the mean SEM (n = 4). The data shown were from a set of
representative experiments. *** (p = 0.0006) and **** (p < 0.0001) indicate the
significant difference between blocked and control studies by using the two-way
ANOVA statistical test (GraphPad Prism 6.0). ns means nonsignificant.
also worth mentioning that Tc1 presents
a
relevant
accumulation in adrenals, a target tissue with high NET density.
To assess whether the moderate heart uptake is specifically
related to NET, we have performed a blockade experiment by
Table 1. Biodistribution of Tc1 in CD1 mice at 2, 30, and 60 min (% ID/g SD)
% ID/g
Coinjection with nonradioactive MIBG
60 min
Organ
2 min
30 min
60 min
Blood
Liver
Intestine
Spleen
Heart
Lung
Kidney
Muscle
Bone
Stomach
Brain
Thyroid
Adrenal glands
Ratio (heart/blood)
Ratio (heart/liver)
Ratio (heart/kidney)
Ratio (heart/lung)
Total excretion (% ID)
3.5 0.7
17.4 7.5
4.5 0.4
1.37 0.08
7.2 1.5
2.7 0.7
67.9 13.7
1.16 0.05
1.41 0.06
1.2 0.4
0.18 0.03
2.1 0.2
6.0 3.1
2.1
0.66 0.03
22.4 8.9
5.7 0.7
0.9 0.5
7.8 1.3
0.9 0.3
72.9 4.5
0.7 0.1
0.6 0.1
1.5 0.7
0.11 0.07
1.3 0.3
—
0.7 0.3
24.8 5.6
6.7 2.4
0.24 0.01
6.3 1.3
0.8 0.4
73.7 22.9
0.5 0.2
0.5 0.1
1.4 0.3
0.10 0.05
1.1 0.5
4.8 3.5
9.0
0.75 0.07
21.98 3.60
6.44 0.62
1.47 0.19
3.89 0.38
1.30 0.30
77.14 5.33
0.31 0.02
0.35 0.02
0.86 0.37
—
—
1.3 0.1
5.2
0.18
0.05
11.8
0.35
0.11
8.6
0.41
0.11
2.8
0.25
0.08
8.9
5.2 1.3
3.0
7.1 0.8
—
3.8 1.1
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B. L. Oliveira et al.
coinjecting the mice with nonradioactive MIBG (1.0 mg/kg). In to decrease kidney and liver uptakes, while maintaining the high
this group of animals, the heart uptake of Tc1 was reduced by persistent cardiac accumulation.
38.2% at 60 min. This result suggests that the heart uptake
mechanism of Tc1 may be related to that of MIBG and is in
agreement with the conclusion drawn from the cell uptake
Acknowledgements
studies.
Aiming to confirm the hypothesis that the Tc1 binding is
This work was supported by the Fundação para a Ciência e
Tecnologia (FCT), Portugal, through project PTDC/QUI-QUI/
121752/2010. M. Morais and B. L. Oliveira thank FCT for Ph.D.
fellowships (SFRH/BD/48066/2008 and SFRH/BD/38753/2007,
respectively). Covidien-Mallinckrodt is acknowledged for the
IsoLink kits and Dr. J. Marçalo for performing the ESI-MS
analyses. The ESI/QITMS was acquired with the support of the
Programa Nacional de Reequipamento Científico of FCT and is
part of RNEM-Rede Nacional de Espectrometria de Massa also
supported by FCT.
specific and mediated by interaction of the guanidine moiety
with the NET in vivo, we have also prepared Tc2, where the
benzylguanidine pharmacophore has been replaced by a
benzylamine group. In this case, we were not expecting a NET-
mediated accumulation of radioactivity in the heart. The data
of the biodistribution studies of Tc2 confirmed our expectation,
and a negligible heart uptake (0.56 0.23% ID/g at 60-min p.i.;
Table SI) was observed for this radioactive probe. This result is
consistent with the fact that Tc1 does not accumulate in the
heart by a simple diffusion mechanism because Tc2, which
displays similar physicochemical properties such as hydrophilicity
and charge, has low accumulation in that organ.
Conflict of interest
The authors did not report any conflict of interest.
Stomach uptake was very low for Tc1, indicating high in vivo
stability of the complex, which was also confirmed by RP-HPLC
analysis of urine and blood samples. However, a high liver
(>24% ID/g) and kidney (>73% ID/g) accumulation was
observed, which are considerably higher than the values found
for [125I]MIBG at 1-h p.i. (liver: 7.97 0.97% ID/g and kidney:
3.21 0.41% ID/g) in the same animal model 50. With the aim
of reducing the high level of radioactivity retention in the kidney,
which is most likely related with tubular reabsorption, we have
performed an additional set of biodistribution experiments in
which Lys was coinjected with Tc1 (Table SI). The
coadministration of cationic amino acids (Lys and Arg) to inhibit
renal accumulation of radiolabeled proteins and peptides is a
well-established procedure, routinely used in the clinical
setting.51,52 Interestingly, coadministration of Lys did not
significantly reduce kidney accumulation of the radioactive
complex (61.45 12.78% ID/g at 60-min p.i. for Tc1 without
coinjection of Lys and 56.19 8.68% ID/g at 60-min p.i. for Tc1
with coinjection of Lys), which seems to indicate that the
electrostatic interaction between the positively charged complex
and the negatively charged surface of the proximal tubule cells is
not a major factor for renal radioactivity retention.
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Supporting Information
Additional supporting information may be found in the online
version of this article at the publisher’s web-site.
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