Biological assessment of novel bisphosphonate-containing 99mTc/Re-organometallic complexes Dedicated to Prof. M.J. Calhorda on the occasion of her 65th birthday.

Article abstract of DOI:10.1016/j.jorganchem.2013.10.038

Bone-seeking radiopharmaceuticals such as radiolabeled bisphosphonates (BPs) bind to bone matrix in areas of increased bone turnover, characteristic of metastization sites, delivering selectively γ radiation or β^- particles for bone imaging or bone-pain palliation, respectively. Recent preclinical studies suggest that nitrogen-containing BPs may have also a direct anti-tumor activity. We have recently introduced a set of ^99mTc-organometallic complexes that allow the simultaneous delivery of γ radiation and BPs to bone tissue. Aimed at finding complexes of the same family with improved bone-seeking properties and adequate tumor cell uptake profile, we have synthesized novel complexes of the type fac-[M(CO) ₃(k³-L)]⁺ (M = ^99mTc/Re, Tc3/Re3-Tc5/Re5) stabilized by bifunctional chelators with different molecular weight, overall charge, (lipo)hydrophilic nature and different positions of BP attachment. Biodistribution studies in normal mice have shown that Tc3, where the BP unit (alendronate) is at position 4 of the pyrazolyl ring, presents the most favorable pharmacokinetic profile with fast blood clearance, high bone uptake (17.1 ± 3.6% I.A./g at 1 h p.i.), and higher bone-to-blood and bone-to-muscle ratios than the gold standard ^99mTc-MDP. Cellular uptake studies in the MDAMB231 metastatic breast cancer cells showed a maximal uptake of 4-6% at 6 h incubation for all complexes. Interestingly, cell fragmentation studies have shown that Tc3 presents the highest accumulation in the cytosol (35%). The promising biological profile of the BP-containing complex Tc3 encourages further research towards evaluation of ¹⁸⁸Re congeners for potential therapeutic applications.

Full text of DOI:10.1016/j.jorganchem.2013.10.038

Journal of Organometallic Chemistry 760 (2014) 197e204
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Biological assessment of novel bisphosphonate-containing 99mTc/Re-
organometallic complexes
,
,
Célia Fernandes a 1, Sofia Monteiro a 1, Patrícia Mendes a, Lurdes Gano a,
Fernanda Marques a, Sandra Casimiro b, Luís Costa b, João D.G. Correia a , Isabel Santos
a Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10 (ao km 139,7), 2695-066 Bobadela LRS,
,
a,
*
*
Portugal
b Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Bone-seeking radiopharmaceuticals such as radiolabeled bisphosphonates (BPs) bind to bone matrix in
Received 4 October 2013
Received in revised form
16 October 2013
areas of increased bone turnover, characteristic of metastization sites, delivering selectively
ꢀ particles for bone imaging or bone-pain palliation, respectively. Recent preclinical studies suggest that
nitrogen-containing BPs may have also a direct anti-tumor activity. We have recently introduced a set of
99mTc-organometallic complexes that allow the simultaneous delivery of
radiation and BPs to bone
g radiation or
b
Accepted 21 October 2013
g
tissue. Aimed at finding complexes of the same family with improved bone-seeking properties and
adequate tumor cell uptake profile, we have synthesized novel complexes of the type fac-[M(CO)3(k3-L)]þ
(M ¼ 99mTc/Re, Tc3/Re3eTc5/Re5) stabilized by bifunctional chelators with different molecular weight,
overall charge, (lipo)hydrophilic nature and different positions of BP attachment. Biodistribution studies
in normal mice have shown that Tc3, where the BP unit (alendronate) is at position 4 of the pyrazolyl
ring, presents the most favorable pharmacokinetic profile with fast blood clearance, high bone uptake
(17.1 ꢁ 3.6% I.A./g at 1 h p.i.), and higher bone-to-blood and bone-to-muscle ratios than the gold standard
99mTc-MDP. Cellular uptake studies in the MDAMB231 metastatic breast cancer cells showed a maximal
uptake of 4e6% at 6 h incubation for all complexes. Interestingly, cell fragmentation studies have shown
that Tc3 presents the highest accumulation in the cytosol (35%). The promising biological profile of the
BP-containing complex Tc3 encourages further research towards evaluation of 188Re congeners for po-
tential therapeutic applications.
Dedicated to Prof. M.J. Calhorda on the
occasion of her 65th birthday.
Keywords:
Bisphosphonates
Bone
Biodistribution
Cell assays
Rhenium
Technetium-99m
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
Using the match pair 186/188Re ( ꢀ-emitters) and 99mTc (
b g-emitter),
skeletal systemic radiotherapy and radiodiagnostics, respectively, is
possible [3,4]. This systemic form of radiotherapy is simple to
administer and complements other treatment options, although it
has not been possible so far to combine with chemotherapy or
bisphosphonates [2]. In vitro analysis of the anti-tumoral effect of
bisphosphonates on human breast cancer cell lines indicated that
they have direct anti-tumor effects and also inhibit breast and
prostate carcinoma tumor cell invasion and cell adhesion to bone
[5e10]. Zoledronic acid combined with external radiation caused a
decrease in cell viability and, for the first time, synergistic cytotoxic
effect was confirmed by isobologram analysis [11]. Moreover, there
is recent preclinical evidence that nitrogen-containing bisphosph-
onates display a certain degree of anti-tumor activity, which has
been attributed to an indirect mechanism (osteolysis inhibition/
decrease in growth factors released from the bone matrix).
Recently, preclinical studies have also indicated that bisphospho-
nates, such as zoledronic acid, may have also a direct anti-tumor
Many patients with cancer develop symptomatic skeletal me-
tastases at an advanced stage of their disease. This situation is often
complicated by pain and present considerable morbidity and
mortality. Besides analgesics, treatment options include external
beam radiotherapy, bisphosphonates, chemotherapy, surgery and
bone-seeking radiopharmaceuticals [1,2]. The latter binds to the
bone matrix in areas of increased bone turnover characteristic of
metastization sites. The result is the selective delivery of beta
particles to areas of amplified osteoblastic activity, and targeting
simultaneously multiple metastases, both symptomatic and
asymptomatic foci, leading to a pain therapeutic/palliative effect.
* Corresponding authors. Tel.: þ351 21 994 62 01.
E-mail addresses: jgalamba@itn.pt, jgalcorr@gmail.com (J.D.G. Correia), isantos@
itn.pt (I. Santos).
1
These authors contributed equally to the manuscript.
0022-328X/$ e see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2013.10.038

198
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
activity [5,6]. Zoledronic acid-mediated apoptosis is associated
with cytochrome c release and consequent caspase activation, a
process that may be initiated by inhibition of key enzymes in
mevalonate pathway leading to impaired prenylation of intracel-
lular proteins including Ras and RhoA, with subsequent inhibition
cases the N-monoalkylated product V was also obtained. After
isolation, this compound has been used as starting material for the
synthesis of L3 after alkylation with bromoacetic acid.
All bifunctional ligands were fully characterized by the usual
analytical techniques in chemistry, including multinuclear NMR
spectroscopy (1H, 13C and 31P), mass spectrometry (ESIeMS), IR
spectroscopy and RP-HLPC. Brought together, the analytical data
confirm the assigned structure.
of chemotaxis induced by SDF-1,
a chemokine significantly
involved in cancer metastasis to bone [7,10].
Taking these results into consideration, we hypothesized that the
combinationofa bisphosphonateunitanda betaemitterradionuclide
(
188Re or 186Re) in the same molecular multi-functional compound,
2.2. Synthesis and characterization of Re3eRe5
would lead to a synergistic effect in pain therapy and/or cancer pro-
gression, compared to the conventional sequential treatment.
Following our previous studies, which aimed at the design of novel
99mTc(I)-labeled bisphosphonates with improved bone-seeking
properties, we have synthesized and biologically evaluated the
complexes Tc1 and Tc2 (Fig. 1), which display high bone uptake,
comparable to the gold standard 99mTc-MDP. Both complexes allowed
The organometallic complexes Re3eRe5 were synthesized and
characterized as non-radioactive surrogates of the corresponding
99mTc congeners aiming to identify their molecular structure by
means of HPLC comparison. The complexes Re3eRe5 were prepared
byligand exchangereactionof the precursor [Re(CO)3(H2O)3]Br with
L1eL3, respectively (Fig. 1). Complex Re3 was synthesized in
refluxing water overnight after pH adjustment (pH 6e7), whereas
the synthesis of Re4 and Re5 was run in a NMR tube at 80 ꢂC using
D2O as reaction solvent and DCl or KOD for pH adjustment (Re4, pH
7; Re5, pH 4). In the latter case, progression of the reactions was
monitored by 1H and 31P NMR spectroscopy. All resulting complexes
were purified by solid-phase extraction using Sep-Pack C18 car-
tridges, following the procedure described in the experimental part.
The complexes were fully characterized by infrared and multi-
nuclear (1H, 13C and 31P) magnetic resonance spectroscopy, mass
spectrometry (ESIeMS) and RP-HPLC. Brought together, the data
the simultaneous deliveryofg radiation and bisphosphonates to bone
tissue in a high specific and selective way. Profiting from this fact,
herein we extended the family of M(I)-labeled bisphosphonates
previously reported in order to include novel complexes (Tc3/Re3e
Tc5/Re5) with various molecular weights, overall charge, (lipo)hy-
drophilicities and differentpositionsof BPattachment (Fig.1). Wewill
also present the biodistribution profile of the 99mTc(I) complexes as
well as the cellular uptake studies in a metastatic breast cancer cell
modelinordertoevaluatetheirpotentialbothasbone-seekingagents
and cytotoxic agents.
obtained were consistent with a
k k
3-N3 or 3-N2O coordination
mode for the pyrazolyl-containing chelator, as found in other
similar Re(I) complexes already described [12e14].
2. Results and discussion
The IR data indicated a facial arrangement of the carbonyl
2.1. Synthesis and characterization of L1eL3
groups in the complexes, with very strong
n (CO) stretching bands
in the range 2033e1820 cmꢀ1, as previously found for related
complexes with the moiety fac-Re(CO)3 [15]. Moreover, the IR
spectrum of Re5 present a very strong band at 1617 cmꢀ1 due to the
The multistep procedure for the synthesis of the bifunctional
ligands L1eL3 with a pendant bisphosphonate unit is displayed in
Scheme 1. Activation of the free carboxylic acid group of the pyr-
azolyl ring in precursor I with TFF/EDC, followed by reaction with
alendronate afforded an intermediate, which after purification by
Sep-Pak C18 cartridge and final Boc deprotection gave L1.
The symmetric bifunctional ligand L2 was obtained in moderate
yield (60%) by N-alkylation of the primary amine in alendronate
with excess of precursor IV under basic conditions. Aiming to
improve the reaction yield we have varied the reaction conditions,
namely time, temperature and stoichiometric ratio. However, in all
n
(C]O) stretching vibration indicating that the oxygen from the
carboxylic acid participates in the coordination sphere (Dn (C]
O) ¼ 68 cmꢀ1 relatively to the free ligand).
The 1H NMR spectra of Re3eRe5 displayed the characteristic
sharp singlet peaks assigned to methyl groups of the pyrazolyl ring
(Re3,
spectra of Re4 and Re5 display also sharp singlet peaks assigned to
H(4) (Re4, 5.95; Re5, 5.99) of the azolyl ring. Owing to the tri-
d 2.14/2.25; Re4, d 2.05/2.14, Re5 d 2.13/2.29). Unlike Re3, the
d
d
dentate coordination mode of L1eL3 the methylenic protons of the
ligand backbone become diastereotopic displaying chemical shifts
and splitting pattern comparable to those found for other previ-
ously reported complexes of the same type [12e14]. Moreover,
unlike Re4 and Re5, the spectrum of Re3 presents also resonances
assigned, respectively to the tertiary amine (
d 6.5) and to the NH2
protons ( 4.97 and 3.64) which became diastereotopic upon
d
d
coordination of the amine group to the metal center.
The 13C NMR spectra presented resonances for all the carbon
nuclei expected for Re3 and Re4.
The 31P NMR spectra of all complexes present only one singlet at
d
19.0 (Re3),
these resonances relatively to the ones of the respective free ligands
(L1, 19.6; L2, 18.7; L3, 18.7) was found, which is a clear indi-
d 23.0 (Re4) and d 18.3 (Re5). No significant shift of
d
d
d
cation that the terminal bisphosphonic acid group is not involved in
the coordination to the metal [16].
2.3. Radiosynthesis of complexes of the type fac-[99mTc(CO)3(k3-L)]
(Tc3, L ¼ L1; Tc4, L ¼ L2; Tc5, L ¼ L3)
The complexes Tc3-Tc5 were prepared in high yield (>95%) and
radiochemical purity (>95%) by reaction of the radioactive
Fig. 1. M(CO)3-complexes containing pendant bisphosphonate units (M ¼ 99mTc, Re).

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
199
Scheme 1. Synthesis of L1eL3. (i) CH2Cl2, EDC, TFF, room temperature, overnight; (ii) THF/H2O, NEt3, reflux, 3 days; (iii) TFA/CH2Cl2, room temperature; (iv) NaOH, H2O reflux; (v)
BrCH2CO2H, H2O, NaOH, reflux, overnight.
precursor fac-[99mTc(CO)3(H2O)3]þ with the corresponding bifunc-
tional ligands L1eL3 (Fig. 1), similarly to what has been described
for Tc1 and Tc2. All complexes were analyzed by RP-HPLC and all
chromatograms present only one species and no precursor or any
other radiochemical species could be detected. The chemical
identity of the radioactive complexes has been ascertained by
Although complexes Tc3eTc5 share the same common BP
pendant unit, we tentatively assign the different HA binding af-
finities to a set of factors that include different length and nature of
the spacer between the BP unit and the metal core, and overall net
charge of the complex. In the case of Tc3, which displays the highest
affinity to HA, the effect of the spacer lengths seems to prevail.
Indeed, that complex has a longer spacer than Tc4 and Tc5, which
seems to reduce potential interferences of the organometallic core
with the BP unit, increasing its affinity to HA. This assumption is
corroborated by the fact that Tc2 presents a spacer with compa-
rable length and similar affinity to HA (ca. 95% at 4 h) [15]. The
higher affinity of Tc5 relatively toTc4 can be rationalized in terms of
overall net charge. Since the former complex is “more negative”
comparing their analytical RP-HPLC g-traces with RP-HPLC UVevis
traces of the rhenium analogs Re3eRe5.
The stability of the radioactive complexes was evaluated upon
incubation of the complexes with phosphate-buffered saline (PBS)
pH 7.4 and human blood serum, at 37 ꢂC. Analysis of the samples by
RP-HPLC demonstrated that all complexes are stable up to 24 h, as
no decomposition products or reoxidation to pertechnetate was
detected.
The (lipo)hydrophilic nature of all complexes was assessed
based on the n-octanol/0.1 M PBS pH 7.4 partition coefficients. The
average Log Po/w values for three independent trials
(Tc2 ¼ ꢀ1.94 ꢁ 0.02 [15]; Tc3 ¼ ꢀ2.08 ꢁ 0.02; Tc4 ¼ ꢀ0.97 ꢁ 0.02;
Tc5 ¼ ꢀ2.18 ꢁ 0.02) highlight the strong hydrophilic nature of all
complexes, which is most likely due to the presence of the pendant
bisphosphonic acid groups. It is also worth mentioning that com-
plex Tc4, stabilized by the symmetric bifunctional ligand L2, dis-
plays the less hydrophilic character.
With the aim of predicting the in vivo bone-targeting properties
of the complexes and to assess their potential as bone imaging
tracers, we have performed hydroxyapatite (HA) adsorption studies
(15 mg HA, 37 ꢂC) with complexes Tc3eTc5. As shown by the
adsorption curves in Fig. 2 all complexes present high and fast
hydroxyapatite binding at 37 ꢂC (>80% after 1 h). It is worth
mentioning that binding of Tc3 to HA is comparable to the gold
standard 99mTc-MDP.
Fig. 2. Adsorption of 99mTc-MDP, Tc3eTc5 onto HA (15 mg) at 37 ꢂC as a function of
incubation time.

200
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
than the latter, its ionic interaction with the Ca2þ cations of the HA
matrix is favored, leading to stronger binding.
Brought together, the results of the HA adsorption studies
prompted us to assess the in vivo properties of all radioactive
complexes.
3. Biodistribution studies
Biodistribution studies of Tc3eTc5 were carried out in normal
Balb/c mice at 1 h, 4 h and 6.5 h post intravenous injection (p.i.) to
assess their pharmacokinetic profile, bone affinity and in vivo
stability. Data from these studies are summarized for the most
relevant organs in Table 1. For comparison, we have also evaluated,
in the same animal model, the tissue distribution profile of 99mTc-
MDP. The tissues uptake was calculated and expressed as a per-
centage of the injected activity per gram of tissue. The whole-
body radioactivity excretion was determined as a percentage of
the total injected activity.
The biodistribution data have demonstrated that all complexes
are cleared from blood via both the hepatic and the renal path-
ways with a moderate total radioactivity excretion. The most
remarkable feature of all compounds is the rapid accumulation
and long retention time in the bone. In spite of the similar tissue
distribution trend, there are significant differences in their phar-
macokinetics. Whereas Tc3 and Tc4 have a fast blood clearance,
especially the former (0.21 ꢁ 0.05% I.A./g blood at 1 h p.i.), Tc5
presents the slowest clearance (6.4 ꢁ 2.1% I.A./g, 1.11 ꢁ 0.06% I.A./g
and 0.62 ꢁ 0.04% I.A./g, at 1, 4 and 6.5 h respectively). The amount
of activity eliminated by either the hepatobiliar or the urinary
routes is also significantly different among the compounds. Tc4
shows the highest accumulation of activity in the hepatobiliar
tract and consequently the slowest total excretion. This finding is
consistent with the less hydrophilic character of this complex.
Complexes Tc3 and Tc4 present a high bone uptake (17.1 ꢁ 3.6%
I.A./g and 17.7 ꢁ 3.0% I.A./g, respectively, at 1 h p.i.), similar to that
previously found for Tc2, which bears the same BP unit
(17.3 ꢁ 6.1% I.D./g), and to that of 99mTc-MDP (17.1 ꢁ 2.4% I.D./g)
[15]. The lowest radioactivity accumulation in the bone was found
for Tc5. All the compounds have a long residence in the bone up to
6.5 h, however, contrasting with 99mTc-MDP, a slight washout was
observed at later time points. The high bone uptake of Tc3 asso-
ciated to the fast clearance from blood stream and soft tissues like
muscle resulted in a significantly higher bone-to-blood and bone-
to-muscle radioactivity ratio (Table 1). Moreover, this complex
shows also a significantly higher bone-to-blood and bone-to-
muscle ratios than Tc2 and 99mTc-MDP, as displayed in Fig. 3.
Furthermore, Tc3 has the fastest overall excretion rate among all
complexes (59.5 ꢁ 4.0% I.A., 67.3 ꢁ 4.1% I.A. and 66.4 ꢁ 2.7% I.A., at
1, 4 and 6.5 h respectively), which is slightly slower than that
previously described for Tc2 (66.8 ꢁ 7.6% I.A., 77.2 ꢁ 0.5% I.A. and
78.2 ꢁ 1.6% I.A., at 1, 4 and 6.5 h respectively) but significantly
higher than that found for 99mTc-MDP (49.1 ꢁ 6.1% I.A., 56.8 ꢁ 0.9%
I.A. and 59.7 ꢁ 1.7% I.A., at 1, 4 and 6.5 h respectively) in the same
animal model [15].
The in vivo stability of Tc3eTc5 was assessed by RP-HLPC
analysis of blood and urine samples collected at sacrifice time.
The radiochromatograms indicated that all complexes are stable
and, as an illustrative example, those obtained for Tc3 are pre-
sented in Fig. 4.
Brought together, the biodistribution studies demonstrate that
all complexes are able to deliver radiation to bone in a very se-
lective way. However, in order to elicit a cytotoxic effect, namely
through inhibition of key enzymes of the mevalonate pathway,
the complexes must cross the cell membrane and be retained in
the cytoplasm in order to interact with the putative cytosolic

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
201
350
300
250
200
150
100
50
Bone/ Blood
Bone/ Muscle
Tc3
99m
Tc-MDP
Tc2
Fig. 5. Cellular uptake of Tc2eTc5 complexes at 37 ꢂC in MDAMB231 breast cancer
cells.
0
Re/99mTc tricarbonyl complexes. The 99mTc-organometallic com-
plexes (Tc3eTc5) where obtained in high yield and radiochemical
purity. The biodistribution studies in normal mice demonstrated
that all complexes are rapidly taken up by the bone with long
residence times. Complex Tc3 presents the most favorable phar-
macokinetic profile with fast blood clearance, high bone uptake and
higher bone-to-blood and bone-to-muscle ratios than the gold
standard 99mTc-MDP. Among all complexes, Tc3 shows the highest
accumulation in the cytosol, an important requisite for inhibition of
key enzymes in the mavelonate pathway, which is related to po-
tential anti-tumor activity. The promising biological properties of
the BP-containing complex Tc3, both in tumor cells and mice, en-
courages further research towards evaluation of its 188Re congener
for bone metastases therapy.
1 h
4 h
6.5 h
1 h
4 h
6.5 h
1 h
4 h
6.5 h
Time (h)
Fig. 3. Bone-to-blood and bone-to-muscle radioactivity ratios of Tc3 in comparison
with Tc2 and 99mTc-MDP in Balb/c mice at different time points p.i.
targets. Therefore, aiming to assess this issue, we have performed
cellular uptake studies in the MDAMB231 breast cancer cells. These
cells were selected taking into consideration their ability to induce
bone metastases. The results of the cellular uptake as a function of
incubation time is presented in Fig. 5. All complexes present a very
similar profile, with a maximal uptake of 4e6% at 6 h incubation,
which is significantly higher than the value found for 99mTc-MDP at
6 h (0.90 ꢁ 0.004%), under the same experimental conditions.
In order to evaluate complex cellular distribution, MDAMB231
cells were exposed to Tc2eTc5 during 6 h at 37 ꢂC, and the cytosol,
membrane, nucleus and cytoskeletal fractions were isolated and
the radioactivity of each fraction counted. The results depicted in
Fig. 6 show that all radioactive complexes accumulate preferen-
tially at the membrane (>50%). More than 20% total activity was
found in the cytosol and nucleus in particular for complexes Tc2
(35%) and Tc3 (44%). The latter complex Tc3 presented the highest
accumulation in the cytosol (35%).
5. Experimental section
5.1. Chemistry
Unless otherwise stated, all chemicals and solvents were of re-
agent grade and used without purification. The compounds 2-(1-
(2-(tert-butoxycarbonyl(2-(tert-butoxycarbonylamino)ethyl)
amino)ethyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid [14], (4-
Amino-1-hydroxybutylidene)bisphosphonic acid trisodium salt
tetrahydrate (Alendronate, ALN) [17], 1-(2-bromoethyl)-3,5-
dimethyl-1H-pyrazole [18] and tert-butyl 2-bromoethylcarbamate
[19] were prepared according to published methods. The purifica-
tion of the bisphosphonate-containing compounds was achieved
by using Sep-Pak C18 cartridges (Waters Co., 12 cc/2 g) precondi-
tioned according to the manufacturer’s protocol and, unless
otherwise stated, eluted using a H2OeMeOH gradient. Elution of
the products started with water, followed by mixtures of MeOHe
Taking into consideration the results from biodistribution
studies, cellular uptake and cellular distribution, Tc3 appeared as
the most promising radioactive bisphosphonate to be further
evaluated for therapeutic purposes.
4. Conclusions
We have synthesized and characterized
bisphosphonate-containing bifunctional chelators and respective
a set of new
Fig. 4. RP-HPLC chromatograms (radiometric detection) of blood and urine from Balb/
c mice injected with Tc3, at 1 h p.i.
Fig. 6. Cellular distribution of Tc2eTc5 in MDAMB231 cells after 6 h at 37 ꢂC.

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C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
H2O (20e100%). The presence of bisphosphonates in the elution
fractions was monitored by TLC (thin-layer chromatography) using
the Dittmer’s reagent as the revelator agent [20].
100% MeOH. Compound III was obtained as a white powder with
58% (49 mg) yield.
1H NMR (D2O,
d (ppm)): 1.24 (9H, s, Boc); 1.29 (9H, s, Boc); 1.75e
The 1H, 13C and 31P 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 and the 31P chemical shifts with external 85%
H3PO4 solution. IR spectra were recorded as KBr pellets on a Bruker,
Tensor 27 spectrometer. Electrospray ionization mass spectrometry
(ESIeMS) was performed at ITN on a QITMS instrument. The
starting material fac-[Re(CO)3(H2O)3]Br was synthesized by the
literature method [21]. Na[99mTcO4] was eluted from a commercial
99Mo/99mTc generator, using 0.9% saline solution. The radioactive
precursor fac-[99mTc(CO)3(H2O)3]þ was prepared using an Iso-
LinkRÒ kit (Covidean/Malinckrodt).
1.83 (4H, m, 2CH2-ALN); 2.15 (3H, s, CH3-pz); 2.21 (3H, s, CH3-pz);
2.65 (2H, t, CH2); 3.05e3.18 (6H, m, CH2); 3.50 (2H, br, CH2), 4.28
(2H, br, CH2). 31P NMR (D2O,
d): 19.44.
5.2.3. Synthesis of 4-(2-(1-(2-(2-aminoethylamino)ethyl)-3,5-
dimethyl-1H-pyrazol-4-yl)acetamido)-1-hydroxybutane-1,1-
diyldiphosphonic acid (L1)
TFA (3 mL) was added to compound III (24 mg, 0.036 mmol)
and the reaction mixture was stirred for 1 h at room temperature.
TFA was removed under vacuum and the crude was purified by
washing it with diethyl ether, giving L1 (quantitative) as a white
powder.
Column chromatography was performed with silica gel 60
(Merck). HPLC analysis of the Re and 99mTc complexes was per-
formed on a PerkineElmer LC pump 200 coupled to a LC 290
tunable UVeVis detector and to a Berthold LB-507A radiometric
detector, using an analytic MachereyeNagel C18 reversed-phase
column (Nucleosil 100e10, 250 ꢃ 4 mm) with a flow rate of
1 mL/min. HPLC solvents consisted of 0.5% CF3COOH in H2O (sol-
vent A) and 0.5% CF3COOH solution in acetonitrile (solvent B),
gradient: t ¼ 0e3 min, 0% eluent B; 3e3.1 min, 0e25% eluent B;
3.1e9 min, 25% eluent B; 9e9.1 min, 25e34% eluent B; 9.1e20 min,
34e100% eluent B; 20e24 min, 100% eluent B; 24e26 min, 100e0%
eluent B; 26e30 min, 0% eluent B. HPLC analysis of L1-L3 was
performed with solvents consisted of 0.1% CF3COOH in H2O (solvent
A) and 0.1% CF3COOH solution in acetonitrile (solvent B). Gradient
for L1-L3 analysis: t ¼ 0e5 min, 0% eluent B; 5e20 min, 5e95%
eluent B; 20e20.1 min, 100% eluent B; 20.1e30 min, 100% eluent B.
Gradient for L3 analysis: t ¼ 0e5 min, 0% eluent B; 5e20 min, 40e
60% eluent B; 20e25 min, 40e60% eluent B; 25e30 min, 100%
eluent B.
1H NMR (D2O,
d (ppm)): 1.60e1.75 (4H, m, 2CH2-ALN); 2.09 (3H,
s, CH3-pz); 2.14 (3H, s, CH3-pz); 3.03 (2H, t, CH2); 3.20 (2H, br, CH2);
3.28 (4H, m, CH2); 3.42 (2H, br, CH2); 4.44 (2H, br, CH2). 31P NMR
(D2O, d d): 173.22 (C]O); 112.55 (C-4pz);
): 19.56. 13C NMR (D2O,
147.68 (C-3pz); 143.47 (C-5pz); 73.30 (C); 46.88 (CH2); 44.92 (CH2);
44.45 (CH2); 40.01 (CH2); 35.54 (CH2); 31.09 (CH2); 29.70 (CH2);
23.40 (CH2); 9.95 (CH3); 8.97 (CH3).
IR (KBr,
n
/cmꢀ1): 1670, 1170, 1057. ESIeMS (þ) (m/z): calc
[M þ H]þ, 472.16; found, 472.2. ESIeMS (ꢀ) (m/z): calcd [M ꢀ H]ꢀ,
470.16; found, 470.2;
5.3. Synthesis of ligands L2 and L3
5.3.1. Synthesis of 4-(2-(3,5-dimethyl-1H-pyrazol-1-yl)
ethylamino)-1-hydroxybutane-1,1-diyldiphosphonic acid (V)
The pH of a solution of alendronate (330 mg, 0.85 mmol) and 1-
(2-bromoethyl)-3,5-dimethyl-1H-pyrazole (346 mg, 1.70 mmol) in
water (3 mL) was adjusted with a 3 M NaOH solution until,
approximately, pH ¼ 12. The reaction mixture was refluxed during
48 h and after that time the mixture was purified by a conditioned
Sep-Pak C18 (Waters, 12 cc, 2 g) cartridge with a gradient from 100%
H2O to 100% MeOH. Compound V was obtained as a white powder
with 32% (107 mg) yield.
5.2. Synthesis of pyrazolyl-diamine ligand (L1) functionalized with
an alendronate unit at the 4-position of the azolyl ring
1H NMR (D2O,
2.12 (3H, s, CH3-pz); 2.66 (2H, t, CH2); 3.02 (2H, t, CH2); 4.05 (2H, t,
CH2); 5.82 (1H, s, H(4)-pz). 31P NMR (D2O, ): 18.69. 13C NMR (D2O,
): 149.4 [C(3/5)pz]; 142.0 [C(3/5)pz]; 105.5 [C(4)pz]; 74.1 (CeOH);
d (ppm)): 1.74 (4H, m, CH2); 2.03 (3H, s, CH3-pz);
5.2.1. 2,3,5,6-Tetrafluorophenyl-2-(1-(2-(tert-butoxycarbonyl-(2-
(tert-butoxycarbonyl-amino)ethyl)amino)ethyl)-3,5-dimethyl-1H-
pyrazol-4-yl)acetate (II)
d
d
To
a
solution of 2-(1-(2-(tert-butoxycarbonyl(2-(tert-butox-
49.2 (CH2); 47.6 (CH2); 45.9 (CH2); 31.7 (CH2); 23.1 (CH2); 12.3
(CH3); 10.2 (CH3). ESIeMS (þ) (m/z): calc [M þ 3Nae2H]þ, 438.2;
found, 438.1. ESIeMS (ꢀ) (m/z): calcd [M ꢀ H]ꢀ, 370.2; found,
370.0; calcd [M ꢀ 2H þ Na]ꢀ, 392.2; found, 392.1.
ycarbonylamino)ethyl)amino)ethyl)-3,5-dimethyl-1H-pyrazol-4-
yl)acetic acid (I) [14] (577 mg, 1.31 mmol) in dry CH2Cl2 and TFF
(430 mg, 2.60 mmol) was added dropwise, at 0 ꢂC, a solution of EDC
(546 mg, 2.85 mmol) in dry CH2Cl2. The reaction mixture was
stirred overnight, at room temperature. The solvent was removed
under vacuum and the crude was washed with water, giving II
(quantitative yield) as a yellow oil.
5.3.2. 4-(Bis(2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)amino)-1-
hydroxybutane-1,1-diyldiphosphonic acid (L2)
Alendronate (330 mg, 0.85 mmol) and compound IV (1.0 g,
5.0 mmol) were dissolved in a water/THF solution (3/1, 8 mL). The
pH of the solution was adjusted to 12 with a solution of NaOH 3 M
and the reaction mixture was refluxed during 4 days. After this time
the mixture was purified by a conditioned Sep-Pak C18 (Waters, 12
cc, 2 g) cartridge with a gradient from 100% H2O to 100% MeOH.
Compound L2 was obtained as a white powder with 60% yield.
1H NMR (CDCl3,
d): 1.37 (9H, s, 3CH3); 1.42 (9H, s, 3CH3); 2.17
(3H, s, CH3-pz); 2.20 (3H, s, CH3-pz); 3. 11 (2H, t, CH2); 3.37 (2H, t,
CH2); 3.51 (2H, t, CH2); 3.66 (2H, s, CH2); 4.09 (2H, m, CH2).
5.2.2. 4-(2-(1-(2-(tert-butoxycarbonyl(2-(tert-
butoxycarbonylamino)ethyl)amino)ethyl)-3,5-dimethyl-1H-
pyrazol-4-yl)acetamido)-1-hydroxybutane-1,1-diyldiphosphonic
acid (III)
To an aqueous solution (3 mL) of alendronate (85 mg,
0.181 mmol) was added NEt3 to adjust the pH to 10. Compound II
(107 mg, 0.181 mmol) was dissolved in dry THF (3 mL) and added
dropwise to the solution of alendronate. The reaction mixture was
stirred for 5 days at room temperature and the pH maintained at 10.
After that time the mixture was purified by a conditioned Sep-Pack
C18 (Waters, 12 cc, 2 g) cartridge with a gradient from 100% H2O to
1H NMR (D2O,
2.10 (6H, s, CH3-pz); 2.54 (2H, t, CH2); 2.78 (4H, t, CH2); 3.95 (4H, t,
CH2); 5.78 (1H, s, H(4)pz). 31P NMR (D2O, ): 18.70. 13C NMR (D2O,
): 149.0 [C(3/5)pz]; 141.7 [C(3/5)pz]; 105.2 [C(4)pz]; 74.2 (CeOH);
54.5 (CH2); 52.7 (CH2); 45.4 (CH2); 32.0 (CH2); 20.9 (CH2); 12.2
d (ppm)): 1.72 (4H, m, CH2); 2.03 (6H, s, CH3-pz);
d
d
(CH3); 10.3 (CH3). IR (KBr, n
/cmꢀ1): 1676, 1551, 1177, 1119, 1055. ESI-
MS (þ) (m/z): calcd [M þ 2NaeH]þ, 538.4; found, 538.3. ESIeMS (ꢀ)
(m/z): calcd [M ꢀ H]ꢀ, 493.4; found, 492.2; calcd [M ꢀ 2H þ Na]ꢀ,
514.4; found, 514.1.

C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
203
5.3.3. Synthesis of 2-((2-(3,5-dimethyl-1H-pyrazol-1-yl)ethyl)(4-
hydroxy-4,4-diphosphonobutyl)amino)acetic acid (L3)
the reaction mixture was heated at 80 ꢂC overnight. 1H NMR (D2O,
(ppm)): 1.80 (4H, m, 2CH2-ALN); 2.13 (3H, s, CH3-pz); 2.29 (3H, s,
d
To a solution of compound V (60 mg, 0.16 mmol) in water (2 mL)
was added bromoacetic acid (27 mg, 0.19 mmol). The pH was
adjusted to approximately 12 with a solution of NaOH 3 M and the
reaction mixture was refluxed overnight. After this time the
mixture was purified by a conditioned Sep-Pak C18 (Waters, 12 cc,
2 g) cartridge with a gradient from 100% H2O to 100% MeOH.
Compound L3 was obtained as a white powder with 70% (42 mg)
yield.
CH3-pz); 2.5e2.3 (1H, m, CH); 3.06-3.3 (2þ2H, m, CH2þ2 HC-H);
3.78e3.73 (1H, m, CH); 4.20 (2H, m, CH2); 5.99 (1H,s, H(4)-pz).
31P NMR (D2O, /cmꢀ1): 1891, 2026 (CO), 1617
d): 18.32. IR (KBr, n
(C]O). Retention time (RP-HPLC): 10.6 min. ESIeMS (þ) (m/z):
calcd [M þ 3Nae2H]þ, 766.5; found, 766.1; ESIeMS (ꢀ) (m/z): calcd
[M ꢀ H]ꢀ, 698.0; found, 698.1; calcd [M þ NaeH]ꢀ, 720.0; found,
720.1.
1H NMR (D2O,
2.10 (3H, s, CH3-pz); 2.72 (2H, t, CH2); 3.04 (2H, t, CH2); 3.21 (2H, s,
CH2); 4.08 (2H, s, CH2); 5.79 (1H, s, H(4)-pz). 31P NMR (D2O,
):
18.69. 13C NMR (D2O,
): 177.8 (C]O); 149.1 [C(3/5)pz]; 141.7 [C(3/
d
(ppm)): 1.72 (4H, m, CH2); 2.00 (3H, s, CH3-pz);
5.5. General procedure for the synthesis of fac-[99mTc(CO)3(k3-L)]
(L ¼ L1eL3): Tc3eTc5
d
d
In a nitrogen-purged glass vial, 100
10ꢀ3 M (Tc4, Tc5) aqueous solution of the appropriate L1eL3 was
added to 900 L of a solution of the organometallic precursor fac-
99mTc(CO)3(H2O)3]þ (10e18 mCi) in saline at pH 7.4 (Tc3,Tc4) or at
m
L of a 10ꢀ4 M (Tc3) or
5)pz]; 105.5 [C(4)pz]; 74.0 (COH); 57.3 (CH2); 55.2 (CH2); 53.1
(CH2); 45.3 (CH2); 31.8 (CH2); 21.2 (CH2); 12.2 (CH3); 10.2 (CH3). IR
m
(KBr,
n
/cmꢀ1): 11345, 1261, 1685. Retention time (RP-HPLC):
[
15.0 min. ESIeMS (þ) (m/z): calcd [M þ H]þ, 518.2; found, 518.0.
ESIeMS (ꢀ) (m/z): calcd [M ꢀ H]ꢀ, 428.3; found, 428.8; calcd
[M ꢀ 3H þ 2Na]ꢀ, 472.2; found, 472.1.
pH 4 (Tc5). The reaction mixture was then heated to 100 ꢂC for 30e
45 min, cooled on an ice bath and then analyzed by RP-HPLC:
Rt ¼ 10.0 min (Tc3); Rt ¼ 15.7 min (Tc4); Rt ¼ 10.6 min (Tc5). The
radioactive complexes were obtained with high yield (>95%) and
high radiochemical purity (>95%).
5.4. General procedure for the synthesis of the Re complexes (Re3e
Re5)
5.6. In vitro studies
fac-[Re(H2O)3(CO)3]Br was reacted with equimolar amounts of
L1eL3 in refluxing water/methanol (L1) or D2O.The pH was previ-
ously adjusted to 6e7 (L1 and L2) or 4 (L3). After 18 h reaction time,
the reaction mixtures were cooled to room temperature and the
solvent removed under vacuum. The desired complexes were ob-
tained in a pure form after purification with Sep-Pak C18 cartridges
(Waters, 12 cc, 2 g) using MeOH/H2O as eluent and a gradient of
100/0, 25/75, 50/50, 100/0. The pure compounds were lyophilized
and obtained as white powders.
5.6.1. Lipophilicity
The lipophilicity of the radioconjugate was evaluated by the
“shakeflask” method [22]. Briefly, 25 mL of the radioconjugates were
added to a mixture of octanol (1 mL) and 0.1 M PBS pH ¼ 7.4 (1 mL),
previously saturated in each other by stirring the mixture. This
mixture was vortexed and centrifuged (3000 rpm, 10 min, room
temperature) to allow phase separation. Aliquots of 25 mL 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 phase by those in the buffer, and the results expressed as
Log Po/w. Log Po/w ¼ ꢀ2.08 ꢁ 0.02 (Tc3); Log Po/w ¼ ꢀ0.97 ꢁ 0.02
(Tc4); Log Po/w ¼ ꢀ2.18 ꢁ 0.02 (Tc5).
5.4.1. fac-[Re(CO)3(
Re3 (11 mg, 55%) was obtained as a white powder after purifi-
cation and lyophilization. 1H NMR (D2O,
(ppm)): 1.67e1.85 (4H,
k
3L1) (Re3)
d
m, 2CH2-ALN); 2.14 (3H, s, CH3-pz); 2.25 (3H, s, CH3-pz); 2.77e2.81
(2H, m, CH2); 3.10 (2H, m, CH2-ALN); 3.33 (2H, s, CH2); 3.40e3.55
(4H, m, 4CH2); 3,64 (1H, m, NH); 3.98e4.07 (2H, m, CH2); 4.33e4.43
5.6.2. Blood serum stability
Complexes Tc3eTc5 (50
blood serum and incubated at 37 ꢂC. After incubation (0, 1, 2 and
4 h), aliquots (50 L) were taken and the proteins precipitated with
ethanol (100 L). The mixture was centrifuged at 3000 rpm for
15 min at 4 ꢂC and the supernatant (protein-free) filtered through a
Millipore filter (0.22 m), and was analyzed by RP-HPLC.
mL) were added to 500 mL of human
(2H, m, CH2); 4.96 (1H, br, NH); 6.50 (1H, br, NH). 13C NMR (D2O,
d):
200.66; 180.41 (C]O); 159.04 [C(3/5)pz]; 149.78 [C(3/5)pz]; 118.83
[C(4)pz]; 80.11; 61.37 (CH2); 55.71 (CH2); 54.50 (CH2); 49.68 (CH2);
48.55 (CH2); 46.89 (CH2); 37.88 (CH2); 37.21 (CH2); 20.45 (CH3pz),
m
m
16.47 (CH3pz). 31P NMR (D2O,
d): 19.04. IR (KBr,
n
/cmꢀ1): 1666 (C]
m
ONH), 1905, 2025 (CO). Retention time (RP-HPLC): 9.87 min. ESIe
MS (ꢀ) (m/z): calcd [M þ H]þ, 740.09; found, 740.10.
5.6.3. Hydroxyapatite (HA) binding
Adsorption of Tc3eTc5 onto HA was accomplished following an
5.4.2. fac-[Re(CO)3(k3L2) (Re4)
adaptation of previously described methods [23e25]. Briefly, 50
of each complex (w27.8 Ci/25 L) were incubated for 0,1, 2 and 4 h
at 37 ꢂC in a water bath with 15 mg of solid HA and 500
L of
phosphate-buffered saline (PBS) solution (pH 7.2). The liquid and
solid phases were separated by centrifugation (7500 rpm/3 min)
mL
Re4 (2.0 mg, 13%) was obtained as a white powder after purifi-
cation and lyophilization. The pH was previously adjusted to 7.0
with DCl and KOD and the reaction mixture heated at 80 ꢂC over-
m
m
m
night. 1H NMR (CD3OD,
d (ppm)): 1.97 (4H, m, 2CH2-ALN); 2.05 (6H,
s, CH3-pz); 2.14 (6H, s, CH3-pz); 3.06 (2H, m, CH2-ALN); 3.29 (2H, m,
and the solid phase washed twice with 500 mL of PBS solution (pH
CH2); 3.58 (2þ2H, m, 4 HC-H); 4.38 (2H, m, CH2); 5.95 (2H, s, H(4)-
7.2). The activity in the liquid and solid phases was determined
using an ionization chamber.
pz). 13C NMR (CD3OD,
d): 199.2; 198.5 (C^O); 149.4 [C(3/5)pz];
141.8 [C(3/5)pz]; 105.0 [C(4)pz]; 54.4(CH2); 52.5 (CH2); 43.7 (CH2);
32.6 (CH2); 20.0 (CH2); 12.3 (CH3); 10.3 (CH3). 31P NMR (CD3OD,
d):
5.7. Biodistribution studies
23.03. IR (KBr, n
/cmꢀ1): 1820, 2020 (CO). Retention time (RP-HPLC):
15.5 min. ESIeMS (þ) (m/z): calcd [M þ H]þ, 765.1; found, 765.3.
The ex-vivo biodistribution of the complexes was evaluated in
groups of 4e5 adult Balb/c female mice (Charles River) weighing
approximately 15 g each. The animals were injected intravenously
ESIeMS (ꢀ) (m/z): calcd [M ꢀ H]ꢀ, 763.1; found, 763.2.
5.4.3. fac-[Re(CO)3(k3L3) (Re5)
with 100 mL (10e32 MBq) of each preparation via the tail vein and
Re5 (3.0 mg, 27%) was obtained as a white powder after puri-
fication and lyophilization. The pH was adjusted to 4 with DCl and
were maintained on normal diet ad libitum. All animal studies were
conducted in accordance with the highest standards of care, as

204
C. Fernandes et al. / Journal of Organometallic Chemistry 760 (2014) 197e204
outlined in European law. Mice were killed by cervical dislocation
at 1 and 4 h after injection. The injected radioactive dose and the
radioactivity remaining in the animal at sacrifice were measured
with a dose calibrator (Capintec, CRC25R). The difference between
the radioactivity in the injected and the sacrificed animal was
assumed to be due to total excretion from whole animal body.
Blood samples were taken by cardiac puncture when the animals
were killed. Tissue samples of the main organs were removed,
weighed and counted using a gamma counter (Berthold, LB211).
Biodistribution results were expressed as the percentage of the
injected activity (I.A.) per gram of tissue. Statistical analysis of the
biodistribution data was done by an analysis of variance with
GraphPad Prism. Student t test was then used to analyze data from
each compound versus the reference compound 99mTc-MDP. The
level of significance was set at 0.05.
cytoskeletal and nuclear fractions were extracted using a
FractionPREPÔ. cell fractionation system (BioVision. USA) and
performed according to the manufacturer’s protocol. The radioac-
tivity of each fraction was counted in a g-counter.
Acknowledgments
This work has been supported by the Fundação para a Ciência e
Tecnologia (FCT) through the project PTDC/QUI-QUI/115712/2009.
Sofia Monteiro thanks FCT for a BI research grant. Dr. Joaquim
Marçalo is acknowledged for the ESIeMS analyses, which were run
on a QITMS instrument acquired with the support of the Programa
Nacional de Reequipamento Científico (Contract REDE/1503/REM/
2005-ITN) of FCT and as part of RNEM e Rede Nacional de Espec-
trometria de Massa.
5.8. In vivo stability
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