Synthesis, metal complexation and biological evaluation of a novel semi-rigid bifunctional chelating agent for 99mTc labelling

Article abstract of DOI:10.1016/j.bmc.2005.12.005

A novel bifunctional chelating agent bearing an aromatic ring has been synthesised and characterised. This ligand formed well-defined oxorhenium complexes. The analogous ^99mTcO-complex was obtained in an excellent yield with high radiochemical purity (>95%). The biodistribution of the ^99mTc-complex after intravenous injection studied in normal rats showed that the activity was excreted mainly via renal-urinary pathway indicating its use for labelling peptides with ^99mTc.

Full text of DOI:10.1016/j.bmc.2005.12.005

Bioorganic & Medicinal Chemistry 14 (2006) 2904–2909
Synthesis, metal complexation and biological evaluation of a
novel semi-rigid bifunctional chelating agent for 99mTc labelling
Julien Le Gal,a Sandra Michaud,a Marie Gressier,a Yvon Coulaisb and Eric Benoista,*
aLaboratoire de Chimie Inorganique, EA 807, Universite´ Paul Sabatier, Bat. IIR1, 118, route de Narbonne, 31062 Toulouse, France
bLaboratoire ꢀTraceurs et traitement de l’imageꢁ, Faculte´ de Me´decine de Purpan, EA 3033, 133, route de Narbonne,
31062 Toulouse, France
Received 28 October 2005; revised 24 November 2005; accepted 2 December 2005
Available online 11 January 2006
Abstract—A novel bifunctional chelating agent bearing an aromatic ring has been synthesised and characterised. This ligand formed
well-defined oxorhenium complexes. The analogous 99mTcO-complex was obtained in an excellent yield with high radiochemical
purity (>95%). The biodistribution of the 99mTc-complex after intravenous injection studied in normal rats showed that the activity
was excreted mainly via renal-urinary pathway indicating its use for labelling peptides with 99mTc.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
high stability chelators for these two nuclides for sub-
sequent use in diagnostic and therapy, respectively.
There is a great current interest in the use of bifunctional
chelating agents (BFCAs) in labelling of peptides and
proteins with technetium-99m and rhenium-186/188
for the development of target-specific radioimaging/
radiotherapeutic agents, respectively.1 The technetium-
99m is the most important radionuclide in diagnostic
nuclear medicine due to its ideal nuclear properties
(T1/2 = 6 h, 140 KeV c emitter, convenient availability
from a commercial generator), while rhenium-186
(T1/2 = 3.78 days, bÀ (91%) and c (9%) emitter) and rhe-
nium-188 (T1/2 = 16.98 h, bÀ (85%) and c (15%) emitter)
are two of the most promising radionuclides for radio-
therapy.1,2 Typically, BFCAs are chelating ligands com-
prising a donor atom set to strongly coordinate the
metal radioisotope and a functional group for attach-
ment to a bio-targeting vector. The ability of a BFCA
to avoid in vivo transchelation or in vivo dissociation
of the radionuclide is crucial. So, an ideal BFCA is
one which is able to form a stable and inert complex
in high yield, under mild conditions, without the forma-
tion of isomers. However, although several designs have
been used in practice with success, BFCAs for techne-
tium and rhenium that meet all these criteria do not exist
yet. Consequently, it is of high priority to develop novel,
It is well known that a semi-rigid structure provides a
significant increase for the stability of their complexes.3
Most of the semi-rigid polydentate ligands are based on
a cycloalkyl ring4 (cyclohexane, cyclopentane. . .) instead
of a benzene skeleton.5 To our knowledge, to include an
aromatic cycle in the chelate ring compared to the cyclo-
alkyl moiety presents two advantages: its ease of func-
tionalisation, which is an essential feature for coupling
the ligand to the target molecule, and the fact that this
ligand system does not lead to the formation of syn/anti
isomers.
In connection with our interest in the preparation of
new substitution-inert technetium and rhenium com-
pounds, we have reported, in previous works,6,7 the syn-
thesis of a new family of nonfunctionalised semi-rigid
Ph-XN2S-type ligands (X = O, N or S) characterised
by the presence of an aromatic cycle in the framework
to favour and stabilise the chelate ring of the corre-
sponding complexes by an entropic effect. The reactivity
of these ligands with technetium-99m and the stability of
the corresponding radiocomplexes were also investigat-
ed. These ligands produced unique and highly stable
mono oxotechnetium complexes in the following order
for the ortho substituent: OH > NH2 > Strt.7
Keywords: Radiopharmaceuticals; Technetium-99m; Rhenium; Bifunc-
tional chelating agent; Metallic complexes.
*
In order to label biomolecules with technetium-99m or
rhenium-186/188 via this kind of ligand, we decided to
Corresponding author. Tel.: +33 5 6155 6104; fax: +33 5 6155
6118; e-mail: benoist@chimie.ups-tlse.fr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.005

J. Le Gal et al. / Bioorg. Med. Chem. 14 (2006) 2904–2909
2905
develop a novel BFCA derived from our best candidate
Ph-ON2S. The functionalisation of multidentate ligands
often needs time-consuming multistep syntheses. So,
the aim of this work was to produce an easily synthesised
BFCA derived from Ph-ON2S and to characterise its suit-
ability to complex Tc and Re. In this paper, we report a
simple and convenient route of such a ligand, their
complexes with rhenium (V) and the in vivo behaviour
in normal rats of the corresponding 99mTcO-complex.
Anyway, neither yield nor experimental or spectroscopic
details were given. Despite the apparent simplicity of this
method, compound 2 has never been obtained from com-
mercially available 3-amino-4-hydroxybenzoic acid, in
this way, with satisfactory yields (40% maximum). So,
an alternative pathway was investigated to prepare this
compound in excellent yield. The synthesis of 2 involved
a two-step reaction procedure. The first step consisted in
protection of the carboxylic acid function of 4-hydroxy-
3-nitrobenzoic acid by a classical acid esterification reac-
tion in ethanol, giving 1. The second step consisted in
reduction of the nitro function of 1 by a palladium on
charcoal-promoted hydrogenation reaction in MeOH.
The synthesis of precursor 2 was achieved with a global
yield of 87%.
2. Results and discussion
2.1. BFCA synthesis
To link to the target molecule, we selected the carbonyl
group since many biomolecules contain amino groups
available for amide bond formation. The synthesis of
compound 5 was performed in seven steps as outlined
in Scheme 1.
Precursor 3 was prepared in three steps from a previous-
ly described protocol.7 After an amide coupling reaction
between ethyl glycinate and N-hydroxysuccinimidyl-2-
(triphenylmethylthio)ethanoate9 in the presence of
Et3N in a THF/DMF mixture, basic hydrolysis of the
ethyl ester function followed by the activation of the
acid function with NHS-DCC afforded the precursor 3
in 82% overall yield. The aromatic core 2 reacted with
intermediate 3 in the presence of DMAP in acetonitrile
to give 4 in 92% yield.
The first key step was the preparation of ethyl 3-amino-4-
hydroxybenzoate 2. Papachristou and coll.8 proposed
a one-step synthesis for the structural isomer ethyl
4-amino-3-hydroxybenzoate by refluxing 4-amino-3-
hydroxybenzoic acid in ethanol in the presence of HCl.
HOOC
NH2
(i)
OH
EtOOC
NO2
OH
EtOOC
NH2
OH
HOOC
NO2
OH
(iii)
(ii)
1
2
O
H2N
TrtS
O
H
N
O
(iv)
(vii)
O
(v)
(vi)
TrtS
ONHS
+
O
ONHS
3
O
EtOOC
NH HN
O
OH
S
Trt
4
(viii)
O
HOOC
NH HN
O
OH
S
Trt
5
Scheme 1. Synthetic route of ligand 5. Reagents and conditions: (i) HClg, EtOH, 40%; (ii) HClg, EtOH, 95%; (iii) H2, Pd/C, MeOH, 92%; (iv) Et3N,
THF/DMF, rt, 98%; (v) NaOH 1 M, MeOH, 50 ꢁC then HCl 6 M, 99%; (vi) NHS/DCC, THF, rt, 85%; (vii) 3, DMAP, 60ꢁ, CH3CN, 92%; (viii)
NaOH 1 M, MeOH, 75ꢁ then HCl 3 M, 63%.

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J. Le Gal et al. / Bioorg. Med. Chem. 14 (2006) 2904–2909
The final step consisted in a basic hydrolysis reaction.
Several classical saponification conditions10 (KOH or
NaOH in alcohol at rt) were attempted to obtain the
carboxylic acid 5, but without success. Recently, Ziessel
reported an alternative method to hydrolyse similar
ethyl esters (ethyl aryl esters bearing amide functions
on the aromatic core) using a large excess of NaOH
(50 equiv) in a mixture of water and THF at reflux.11
According to this methodology, we have performed
the saponification by heating compound 4 in the pres-
ence of an excess of NaOH using methanol or THF/
MeOH mixture as a solvent. The progress of the reac-
tion, followed by TLC, showed that a temperature
P75 ꢁC is required. Under this temperature, the sapon-
ification only occurred partially. After 4 h at 75 ꢁC in
methanol, all the starting material 4 was consumed.
Controlled acidification then provided the expected
acid 5, which was isolated pure by column chromatog-
raphy with an acceptable yield (63%). Analytical stud-
ies indicate, without ambiguity, formation of the
expected carboxylic acid 5. However, we have noticed
the formation of by-products as responsible for the sig-
nificant decrease of the isolated yield. 2-(Triphenyl-
methylthio)ethanoic acid, resulting in the break of the
non-aromatic amide bond of compound 4 during
saponification step, was isolated in 10% yield and
clearly identified by proton NMR.
ture. Under these conditions, analytically pure complex
7 was obtained in 69% yield (Scheme 2).
These two oxorhenium complexes were unambiguously
identified by conventional analytical techniques and
their spectroscopic characteristics were similar to those
obtained for nonfunctionalised complexes,7 that is the
complexes exhibit a distorted square pyramidal geome-
try with yl-oxygen in the apical position. Briefly, IR
spectra showed an intense band attributable to the
Re@O stretching vibration14 at 957 and 960 cmÀ1 for
complexes 6 and 7, respectively. Methylene protons of
the tetradentate ligand framework were found to be dia-
stereotopic (AB pattern) with coupling constants in the
range of 17–18.5 Hz. According to the literature,13 we
assigned the downfield signal of both AB patterns to
the endo protons (syn to the Re@O group) and the
upfield signals to the exo ones (anti to the Re@O group).
Finally, negative-ion electrospray of each complex
presents two prominent ion peaks with an isotope distri-
bution pattern consistent with the monomeric anion.
As expected, this structural study confirmed the pres-
ence of a unique isomer with respect to the carbonyl
function. Moreover, the carboxyl group does not coor-
dinate to the rhenium core and thus it could be used
for the coupling to a biomolecule.
The BFCA 5 was performed in seven steps with an over-
all yield of 42%, is stable at room temperature and does
not require any particular condition for long-term
storage.
2.3. 99mTc radiolabelling and biodistribution in rats
Trityl deprotection and 99mTc-labelling of the ligand 5
were performed in situ in a methanol/buffer solution,
pH 8.6 (1/4: v/v), by direct reduction of sodium pertechne-
tate in the presence of an excess of tin chloride at 40 ꢁC
during 30 min (Scheme 2). Under these mild conditions,
the labelling reaction was quantitative and only one neg-
atively charged 99mTcO-product was observed. Purifica-
tion of 99mTcO-complex 8 was accomplished by C-18
reverse-phase HPLC and resulted in only one component
with an excellent radiochemical purity (>95%).
2.2. Rhenium chemistry
The oxorhenium complex 6 was synthesised in good
yield using a ligand exchange reaction with our ligand
12
5 and a slight excess of labile ReO(PPh3)2Cl3 in the
presence of diisopropylamine as deprotonating agent
(Scheme 2). In these conditions, the cleavage of trityl
group was accomplished during the coordination of
the ligand to the ReO3+ core and this is in agreement
with the acidic contribution of the metal in the mecha-
nism of sulfur detritylation.13 The presence of diisopro-
pylammonium chloride as a persistent contaminant,
even after column chromatography purification, sug-
gested the replacement of the countercation with larger
PPh4+. The exchange reactions were performed in
dichloromethane/methanol solutions at room tempera-
Preliminary biological evaluation of HPLC-isolated
complex 8 was performed in healthy rats at 5 and
30 min pi. Complex 8 showed a reasonably high liver up-
take (14% ID at 5 min post-injection), but was preferen-
tially eliminated via the renal-urinary excretion route, as
revealed by the 37% ID in urine at 30 min post-injection
(Table 1), indicating the hydrophilic character of the
radiocomplex. The higher concentration of radioactivity
1) ReOCl3(PPh3)2, iPr2NH, MeOH
O
N
O
O
2) PPh4Cl, MeOH/CH2Cl2
C
HOOC
N
S
O
O
M
HOOC
NH HN
O
OH
S
NaTcO4, SnCl2,
Buffer pH 8.6/MeOH
Trt
6, M = Re, C = iPr2NH2
7, M = Re, C = PPh4
8, M = Tc, C = Na
5
Scheme 2. Preparation of the oxorhenium and oxotechnetium complexes.

J. Le Gal et al. / Bioorg. Med. Chem. 14 (2006) 2904–2909
2907
Table 1. Biodistribution data (% of ID/organ)a of the 99mTcO-complex
8 in normal rats at different time points (pi)
um(VII) oxide, purchased from Aldrich Chem. Co,
was converted to ReOCl3(PPh3)2 according to published
protocols.12 The intermediate 3 has been prepared as
described previously.7 NMR spectra were recorded on
5 min
30 min
Blood
Brain
Heart
29.54 0.09
0.06 0.01
0.41 0.03
4.53 0.17
25.29 2.48
13.91 1.37
0.79 0.12
1.17 0.17
0.20 0.05
0.58 0.20
3.07 1.18
6.55 0.45
0.02 0.01
0.13 0.01
15.74 2.12
7.26 2.34
4.7 0.83
a
Bruker AC 250 (62.896 MHz for 13C and
1
250.133 MHz for H) or 400 (100.63 MHz for 13C and
400.133 MHz for H). Chemical shifts are indicated in
1
Intestine
Kidneys
Liver
d values (ppm) downfield from internal TMS, and cou-
pling constants (J) are given in Hertz (Hz). Infrared
spectra were recorded as KBr pellets on a Bruker Vector
22 spectrophotometer in the range 4000–400 cmÀ1. Neg-
ative electrospray or DCI-Mass spectra were obtained
on a NERMAG R 10-10 mass spectrometer. Microanal-
ysis was performed by the microanalytical department
Lung
0.37 0.04
0.57 0.08
0.05 0.01
0.51 0.28
37.15 10.42
Pancreas
Spleen
Stomach
Urine
a Values represent means SD (n = 3).
´
of the Ecole Nationale Superieure de Chimie de Tou-
louse. HPLC analysis and purification were achieved
on a Waters 600E gradient chromatography with a
Waters Lambda Max UV detector, a SAIP radioactivity
detector and an ICS dual integrator for effluent moni-
toring and a Satisfaction RP18AB column (5 lm,
125 · 4.6 mm) using MeOH–H2O–TFA: 55/45/0.1 as
eluent (flow rate of 1 mL/min).
was measured in kidneys at all times studied (9.74,
2.89% ID/g).
The rapid clearance of this compound from the blood-
stream, reflected in the low blood activity, indicated its
high stability against exchange reactions with blood
proteins and no specific uptake or long-term retention
in organs or tissues. The biodistribution studies also
indicated that little, if any, in vivo decomposition of
complex 8 occurred. The minimal activity accumulation
in the stomach (0.51 0.28% ID/organ at 30 min) and in
spleen (0À.05 0.01% ID/organ at 30 min) indicated that
4.1. Ethyl 4-hydroxy-3-nitrobenzoate 1
A solution of 4-hydroxy-3-nitrobenzoic acid (5.00 g,
27.3 mmol) in freshly distilled ethanol (150 mL) was satu-
rated in HClg. The mixture was stirred at room tempera-
ture overnight and then the solvent was removed under
reducedpressuretogive1asayellowpowder(5.48 g,95%).
99mTcO4 was not produced in relevant amount during
biodistribution.15 The observed stability provides
important evidence that this ligand system is capable
of stabilising 99mTc even under in vivo conditions.
1H NMR (250 MHz, CDCl3) dH (ppm): 1.40 (t, 3H,
J = 7.1 Hz, CH3), 4.38 (q, 2H, J = 7.1 Hz, OCH2), 7.20
(d, 1H, J = 8.8 Hz, HAr), 8.22 (dd, 1H, J = 2.1 and
8.8 Hz, HAr), 8.80 (d, 1H, J = 2.1 Hz, HAr), 10.88 (s,
1H, OH); 13C {1H} NMR (62.9 MHz, CDCl3) dC
(ppm): 14.3 (CH3), 61.7 (OCH2), 120.3, 127.3, 137.8
(3CHAr), 123.2, 133.2, 158.1 (3CAr), 164.4 (CO); MS
(DCI/NH3): 229 [M+NH4+].
3. Conclusion
In short, the synthetic protocol described herein provides
an easy access to an original BFCA including an
aromatic cycle in the chelate ring, in seven steps with
an overall yield of 42%, the rate-limiting step being the
final hydrolysis step. This compound produced well-de-
fined ReO complexes. Moreover, this BFCA formed a
stable and inert 99mTcO-complex in high yield, under
mild conditions, without the formation of isomers. In
vivo study demonstrates the high stability of the radio-
complex and shows that this chelate is efficiently cleared
to a great extent from the bloodstream via the renal-uri-
nary excretion route and to a less extent via the hepatob-
iliary pathway. No specific uptake or retention in the
kidneysorotherorganswasobserved. Allthesesignificant
results justify its use for labelling peptides with 99mTc.
Development of new target-specific radioimaging agents
with this new kind of semi-rigid BCFA is currently under-
way and the results will be disclosed in the near future.
4.2. Ethyl 3-amino-4-hydroxybenzoate 2
Catalytic hydrogenation of 1 (5.00 g, 23.7 mmol) in
methanol (250 mL) over 10% Pd/C (20% w/w, 1.25 g)
was carried out at atmospheric pressure. The mixture
was stirred for 2 h, then the catalyst was filtered off
through Celite and the solvent was removed under
reduced pressure to give 2 as a grey solid (3.92 g, 92%).
1H NMR (250 MHz, MeOD) dH (ppm): 1.33 (t, 3H,
J = 7.1 Hz, CH3), 4.27 (q, 2H, J = 7.1 Hz, OCH2), 6.72
(d, 1H, J = 8.3 Hz, HAr), 7.30 (dd, 1H, J = 2.1 and
8.3 Hz, HAr), 7.40 (d, J = 2.1 Hz, HAr); 13C {1H}
NMR (62.9 MHz, MeOD) dC (ppm): 14.9 (CH3), 61.6
(OCH2), 114.7, 118.0, 122.5 (3CHAr), 122.9, 136.4,
151.3 (3CAr), 168.8 (CO); MS (DCI/NH3): 182
[M+H+], 199 [M+NH4+].
4. Experimental
4.3. Ethyl 3{2-[(triphenylmethylsulfanyl)methylcarbon-
ylamino]ethanamido}-4-hydroxybenzoate 4
All chemicals were of the highest purity commercially
available and all solvents were freshly distilled by stan-
dard methods before use. Column chromatography
was carried out using ꢀgravityꢁ silica (Merck). Rheni-
To a solution of 2 (0.50 g, 2.76 mmol) and 3 (1.35 g,
2.76 mmol) in acetonitrile (40 mL) was added DMAP

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J. Le Gal et al. / Bioorg. Med. Chem. 14 (2006) 2904–2909
(0.34 g, 2.76 mmol). The solution was heated at 60 ꢁC,
under nitrogen, for 3 h. After cooling, the product was
left to precipitate overnight at À18 ꢁC. After filtration,
the precipitate was washed with cold acetonitrile and
then purified by column chromatography on silica gel
(CHCl3 then CHCl3–MeOH 95/5) to afford 4 as a white
powder (1.41 g, 92%).
1H RMN (400 MHz, MeOD): dH = 1.29 (m, 12H, CH3),
3.45 (m, 2H, CHN), 3.75 (d, 1H, J = 17.2 Hz, CH2S),
4.08 (d, 1H, J = 17.2 Hz, CH2S), 4.49 (d, 1H,
J = 18.4 Hz, CH2N), 5.44 (d, 1H, J = 18.4 Hz, CH2N),
7.04 (d, 1H, J = 8.2 Hz, HAr), 6.66 (dd, 1H, J = 8.2
and 1.8 Hz, HAr), 8.90 (m, 1H, HAr); 13C{1H} NMR
(100.6 MHz, MeOD): dC = 8.0 (6CH3), 39.9 (CH2S),
46.8 (2CHN), 60.6 (CH2N), 114.5, 119.5 (2CHAr),
121.4 (CAr), 126.1 (CHAr), 140.2, 169.4 (2 CAr), 174.1,
187.2, 194.2 (3CO); MS (ESÀ), m/z (%): 481 (60), 483
1H NMR (250 MHz, DMSO-d6) dH (ppm): 1.28 (t, 3H,
J = 7.1 Hz, CH3), 2.87 (s, 2H, CH2S), 3.86 (d, 2H,
J = 5.6 Hz, CH2N), 4.25 (q, 2H, J = 7.1 Hz, OCH2),
6.94 (d, 1H, J = 8.3 Hz, HAr), 7.30 (m, 15H, HAr Trt),
7.58 (dd, 1H, J = 1.8 and 8.3 Hz, HAr), 8.35 (s, 1H,
NH), 8.53 (d, 1H, J = 1.8 Hz, HAr), 9.25 (s, 1H, NH),
10.87 (s, 1H, OH); 13C {1H} NMR (100.6 MHz, CDCl3)
dC(ppm): 14.6 (CH3), 35.7 (CH2S), 44.6 (CH2N), 61.2
(OCH2), 68.3 (CTrt), 118.6 (CHAr), 122.6 (CAr), 124.1
(CHAr), 125.5 (CAr), 128.6 (CHAr), 127.4, 128.5, 129.6
(15CHAr Trt), 143.9 (3CAr Trt), 152.6 (CAr), 166.5,
168.3, 170.3 (3CO); MS (DCI/NH3): 555 [M+H+], 572
[M+NH4+].
(100) [MÀ]; IR (KBr): mRe@O = 960 cmÀ1
.
4.6. [PPh4][ReO{(COOH)Ph-ON2S}] 7
Compound 6 (128.5 mg, 0.22 mmol) and tetraphenyl-
phosphonium chloride (89.2 mg, 0.24 mmol) were dis-
solved in a mixture of MeOH (15 mL) and CH2Cl2
(15 mL). After 30 min under stirring, the solvent was
removed and the crude mixture was purified by column
chromatography on silica gel (eluent: CH2Cl2–MeOH,
95/5 then 90/10) to yield the complex 7 as an orange
powder (125.0 mg, yield = 69%).
4.4. 3-{2-[(triphenylmethylsulfanyl)methylcarbonylamino]-
ethanamido}-4-hydroxybenzoic acid 5
1H RMN (400 MHz, CDCl3): dH = 3.75 (d, 1H,
J = 17.1 Hz, CH2S), 4.05 (d, 1H, J = 17.1 Hz, CH2S),
4.41 (d, 1H, J = 18.3 Hz, CH2N), 5.39 (d, 1H,
J = 18.3 Hz, CH2N), 6.97 (d, 1H, J = 8.3 Hz, HAr),
7.51 (m, 9H, HAr + HAr PPh4), 7.68 (m, 8H, HAr
PPh4), 7.82 (m, 4H, HAr PPh4), 8.83 (m, 1H, HAr);
13C{1H} NMR (100.6 MHz, CDCl3): dC = 40.1
(CH2S), 61.0 (CH2N), 116.9, 118.0, 119.9 (3CHAr),
115.2 (4CAr PPh4), 130.5, 130.7, 134.2, 134.3, 135.7,
135.8 (20CHAr PPh4), 141.0, 171.5 (2CAr), 175.3,
186.5, 193.3 (3CO); MS (ESÀ): m/z (%) 481 (60), 483
To a solution of 4 (2.00 g, 3.61 mmol) in MeOH
(180 mL) was added
a solution of 1 N NaOH
(140 mL). After 4 h at 75 ꢁC, the mixture was cooled
and 3 N HCl was added until the solution became acid-
ic. The solution was concentrated (to eliminate the
MeOH) and then extracted with AcOEt (4 · 60 mL).
The organic layer was separated, dried over sodium sul-
fate, filtered off and concentrated to dryness under re-
duce pressure. The residue was purified by column
chromatography on silica gel (CH2Cl2–AcOEt 6/4 then
CH2Cl2–MeOH 95/5) to give 5 as a white powder
(1.20 g, 63%).
(100) [MÀ]; IR (KBr): mC@O = 1644, 1635, 1610 cmÀ1
Re@O = 957 cmÀ1. Found: C, 51.19; H, 3.52; N, 3.24;
C35H28N2O6PReS requires C, 51.15; H, 3.43; N, 3.41.
,
m
1H NMR (400 MHz, DMSO-d6) dH (ppm): 2.89 (s,
2H, CH2S), 3.85 (d, 2H, J = 5.4 Hz, CH2N), 6.92 (d,
1H, J = 8.4 Hz, HAr), 7.33 (m, 16H, HAr + 15HAr
Trt), 7.55 (d, 1H, J = 8.4 Hz, HAr), 8.35 (s, 1H, NH),
8.49 (s, 1H, NH), 9.23 (s, 1H, OH); 13C {1H} NMR
(100.6 MHz, DMSO-d6) dC (ppm): 36.5 (CH2S), 43.9
(CH2N), 66.7 (CTrt), 115.4 (CHAr), 121.9 (CAr),
123.5 (CHAr), 126.4 (CAr), 127.1 (CHAr), 127.5,
128.8, 129.8 (15CHAr Trt), 144.7 (3CAr Trt), 151.1
(CAr), 168.2, 168.6 (3CO); MS (DCI/NH3): 527
[M+H+], 544 [M+NH4+]; IR (KBr): mC@O = 1683,
1652, 1602 cmÀ1; elemental analysis found C, 68.37;
H, 5.05; N, 4.96% C30H26N2O5S requires C, 68.42;
H, 4.98; N, 5.32%.
4.7. 99mTc labelling
Ligand 5 (100 lL of a freshly prepared stock methanolic
solution of 1 mg/mL of 5) was added to a buffer solution
pH = 8.6 (200 lL). Successively were added SnCl2Æ2H2O
(75 lL of a freshly prepared solution of 9 mg of SnCl2Æ
2H2O in a mixture of methanol (4 mL) and HCl concen-
trated (0.5 lL)) and 99mTc-pertechnetate solution gener-
ator eluate (100 lL, 74 MBq). The vial was sealed with a
Teflon-lined cap and the mixture was heated at 40 ꢁC for
30 minutes. After cooling, the resulting complex was
analysed and purified with the HPLC system described
before. The retention time of 8 was 3.88 min.
4.8. Biodistribution in healthy rats
4.5. [iPrNH2][ReO{(COOH)Ph-ON2S}] 6
All experiments were carried out in compliance with
French laws relating to the conduct of animal
experimentation.
To
5 (157.8 mg, 0.3 mmol) and diisopropylamine
(1.68 mL, 0.12 mmol) dissolved in dry methanol (40
mL) was added ReOCl3(PPh3)2 (324.5 mg, 0.39 mmol).
After refluxing for 4 h, the solution was cooled, filtered
and then evaporated to dryness. The residue was puri-
fied by column chromatography on silica gel (eluent:
CHCl3–MeOH: 95/15 then 90/10) to yield the complex
6 as a dark red powder (155 mg, 88%).
Before being used in the animal studies, purified com-
plex 8 solution was filtered through a 0.22 lm sterile fil-
ter (Milliporeꢂ) and diluted with sterile saline solutions.
Male Wistar rats, about 400 g and anaesthetised with
Nesdonalꢂ, were sacrificed 5 and 30 min post-injection

J. Le Gal et al. / Bioorg. Med. Chem. 14 (2006) 2904–2909
2909
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(pi) (n = 3) after an intra-jugular injection of 300 lL of
the diluted tracer solution. The organs of interest (liver,
spleen, heart, lungs, kidneys and brain) were dissected,
weighed and their radioactivity was measured in a Pack-
ard autogamma counter. Results, expressed as percent-
age of injected dose per organ (% ID/organ), are
summarized in Table 1. The percentage radioactivity
in the blood was calculated assuming that the whole-
blood volume was 7% of the body weight.
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