Rhenium and technetium tricarbonyl complexes anchored by 5-HT1A receptor-binding ligands containing P,O/N donor atom sets

Article abstract of DOI:10.1016/j.jorganchem.2004.09.051

The (2-methoxyphenyl)piperazine pharmacophore, a part of the WAY 100635 structure, has been functionalized with phosphinoarylbenzylamide or phosphinoarylbenzylamine chelator groups using propylene or hexylene alkyl chains as linkers (L2-L4). These heterofunctionalized phosphines bearing an arylpiperazine moiety have been used to stabilize rhenium tricarbonyl complexes of the type [Re(CO)₃Br(κ²-L)] (4, L = L2; 5, L = L3; 6, L = L4), which have been fully characterized, including by X-ray crystallographic analysis in the case of compounds 4 and 5. These monomeric complexes are six-coordinate, displaying a distorted octahedral coordination geometry with a facial arrangement of the carbonyl groups. The other three remaining positions are occupied by a bromide and by the bidentate heterofunctionalized phosphine, which coordinates through the phosphorus and the oxygen atom or through the phosphorus and the nitrogen atom in 4 and 5, respectively. The ^99mTc complexes (3a-6a) were also prepared and their characterization established by comparative HPLC, using the Re complexes as surrogates. The in vitro binding affinity for the 5HT_1A receptor subtype and the selectivity against the 5HT_2A receptors for the rhenium complexes were determined. Compound 3 is the only one which presents a reasonable affinity and selectively towards 5HT_1A (IC₅₀ = 20 nM) and 5HT_2A (IC₅₀ = 4680 nM) receptors, respectively. When the spacer length between the chelate unit and receptor binding domain increased and/or the amide group in the chelator was replaced by a secondary amine unacceptable affinity values for 5HT_1A receptors (IC ₅₀ = 200-1100 nM) and lost of selectivity were observed.

Full text of DOI:10.1016/j.jorganchem.2004.09.051

Journal of Organometallic Chemistry 689 (2004) 4811–4819
www.elsevier.com/locate/jorganchem
Rhenium and technetium tricarbonyl complexes anchored by 5-HT1A
receptor-binding ligands containing P,O/N donor atom sets
a
Elisa Palma , Joa˜o D.G. Correia , Angela Domingos , Isabel Santos
a
a
a,
*
^
,
b
Roger Alberto , Hartmut Spies
c
a
´ ´
Departamento de Quımica, ITN, Estrada Nacional 10, 2686-953 Sacavem Codex, Portugal
b
Institute of Inorganic Chemistry, University of Zu¨rich, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland
Institute of Bioinorganic & Radiopharmaceutical Chemistry, Forschungszentrum Rossendorf, D-01314 Dresden, Germany
c
Received 23 August 2004; accepted 21 September 2004
Abstract
The (2-methoxyphenyl)piperazine pharmacophore, a part of the WAY 100635 structure, has been functionalized with phos-
phinoarylbenzylamide or phosphinoarylbenzylamine chelator groups using propylene or hexylene alkyl chains as linkers (L2–
L4). These heterofunctionalized phosphines bearing an arylpiperazine moiety have been used to stabilize rhenium tricarbonyl com-
plexes of the type [Re(CO)3Br(j2-L)] (4, L = L2; 5, L = L3; 6, L = L4), which have been fully characterized, including by X-ray crys-
tallographic analysis in the case of compounds 4 and 5. These monomeric complexes are six-coordinate, displaying a distorted
octahedral coordination geometry with a facial arrangement of the carbonyl groups. The other three remaining positions are occu-
pied by a bromide and by the bidentate heterofunctionalized phosphine, which coordinates through the phosphorus and the oxygen
atom or through the phosphorus and the nitrogen atom in 4 and 5, respectively. The 99mTc complexes (3a–6a) were also prepared
and their characterization established by comparative HPLC, using the Re complexes as surrogates. The in vitro binding affinity for
the 5HT1A receptor subtype and the selectivity against the 5HT2A receptors for the rhenium complexes were determined. Compound
3 is the only one which presents a reasonable affinity and selectively towards 5HT1A (IC50 = 20 nM) and 5HT2A (IC50 = 4680 nM)
receptors, respectively. When the spacer length between the chelate unit and receptor binding domain increased and/or the amide
group in the chelator was replaced by a secondary amine unacceptable affinity values for 5HT1A receptors (IC50 = 200–1100 nM)
and lost of selectivity were observed.
ꢀ 2004 Elsevier B.V. All rights reserved.
Keywords: Rhenium; Technetium; Phosphine; Radiopharmaceuticals; 5HT1A receptor-ligands
1. Introduction
attracted considerable attention in the past years [1].
This interest arises mainly from the fact that several neu-
rological diseases are directly related with changes in the
density of specific receptors, which in the past could only
be detected by post mortem binding studies [1]. The 5-
HT1A subtype of receptors for the neurotransmitter
serotonin is involved in important physiological proc-
esses in the human brain, and changes in their number
are associated with several neurological diseases, such
as Alzheimer disease, schizophrenia, and depression
[2]. Despite the great variety of 5-HT1A radioligands
The search for new radioligands as diagnostic tools
for the visualisation and quantification of neurorecep-
tors in the central nervous system (CNS), either by pos-
itron emission tomography (PET) or single-photon
emission tomography (SPECT), is a subject that has
*
Corresponding author. Tel.: +351 21 994 62 01; fax: + 351 21 994
14 55.
E-mail address: isantos@itn.mces.pt (I. Santos).
0022-328X/$ - see front matter ꢀ 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2004.09.051

4812
E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
with positron-emitting radionuclides for PET imaging,
such as the successful radiopharmaceuticals [carbo-
nyl-11C] WAY-100635 (WAY) and p-[18F]MPPF, there
is still a scope for the development of technetium-
labelled 5-HT1A ligands for SPECT imaging [1,2]. In
which concerns the development of 99mTc-based imag-
ing agents selective for CNS receptors, [99mTc]TRO-
DAT-1 represents up to now the most successful
development for imaging DAT sites [3]. Since the intro-
duction of the low-valent aquo-carbonyl complex fac-
spacer lengths between the chelator unit and the recep-
tor binding domain (L2–L4). We also describe the syn-
thesis and characterization of tricarbonyl complexes of
the type [M(CO)3Br(j2-L)] (M = 99mTc, L = L1 (3a);
M = Re, 99mTc and L = L2 (4, 4a), L = L3 (5, 5a),
L = L4 (6, 6a)) and the in vitro binding affinity of the
Re complexes for 5-HT1A and 5-HT2A receptors, cf.
Scheme 1.
[
99mTc(OH2)3(CO)3]+ as a versatile precursor for the
2. Materials and methods
labelling of biomolecules, a number of biologically
active peptides and CNS receptor ligands have been la-
belled making use of different bifunctional chelating
agents (BFCA) [4–15]. Despite the recent advances in
this area, further achievements still rely on the develop-
ment of new 99mTc-organometallic complexes with ade-
quate in vitro and in vivo behaviour, in order to obtain
radiopharmaceuticals that fulfil the minimum requisites
for CNS receptor imaging.
Recently, we have introduced several heterofunction-
alized phosphines, which are able to stabilize Re and Tc
in low and intermediate oxidation states [11,12,16–19].
For rhenium(I) we have described the complexes [Re-
(CO)3Br(j2-H2PNO)] (1, H2PNO = N-(2-hydroxyethyl)-
2-(diphenylphosphanyl)benzamide), [Re(CO)3(j3-PN2)]
(2, HPN2 = N-(2-aminoethyl)-2-(diphenylphosphanyl)
benzamide), and [Re(CO)3Br(j2-L1)] (3) (L1 = N-[4-(3-
aminopropyl)-1-(2-methoxyphenyl)piperazine]-2-(diphe-
nylphosphanyl)-benzamide), which are the first
examples of fac-[Re(CO)3]+ anchored by heterofunc-
tionalized phosphines containing PN2 and PNO donor
atom sets [11,12].
2.1. General procedures
All Chemicals and solvents were of reagent grade and
were used without further purification. N-[2-(diphenyl-
phosphanyl)benzoyloxy]succinimide, (NEt4)2[ReBr3(CO)3],
N -[4-(3-amino-propyl)-1-(2-methoxyphenyl)piperazine]-2-
(diphenylphosphanyl)benzamide (L1), 4-(6-amino-
hexyl)-1-(2-methoxyphenyl)piperazine, fac-[Re(CO)3Br
(j2-L1)] (3) and fac-[M(OH2)3(CO)3]+ (M = Re, 99mTc)
were prepared according to published methods
1
[4,11,16–19]. H and 31P NMR spectra were recorded
1
on a Varian Unity 300 MHz spectrometer; H chemical
shifts were referenced with the residual solvent reso-
nances relatively to tetramethylsilane and the 31P
NMR chemical shifts with external 85% H3PO4 solu-
tion. NMR spectra were run in CDCl3 and CD3CN.
IR spectra were recorded as KBr pellets on a Perkin–
Elmer 577 spectrometer. Carbon, hydrogen and nitrogen
analysis were performed on a Perkin–Elmer automatic
analyser. Na[99mTcO4] was eluted from a 99Mo/99mTc
generator using 0.9% saline. HPLC analyses were per-
formed on two different HPLC systems: (a) Merck–
Hitachi L-7000-system equipped with an L-7400 tunable
absorption detector, a Berthold LB-508 radiometric
detector; (b) Perkin–Elmer LC pump 200 (binary) cou-
pled to a LC 290 tunable UV/Vis detector, a Berthold
LB-507A radiometric detector. For the radiochemical
analyses two different Macherey–Nagel C-18 reversed-
phase columns were used: nucleosil (5 lm, 250 · 3 mm
– flow rate: 0.5 ml/min) and nucleosil (10 lm, 250 · 4
mm – flow rate 1 ml/min). HPLC solvents consisted of
0.1% CF3COOH in H2O (solvent A) and methanol (sol-
vent B). The HPLC system started with 100% of A from
0 to 3 min. The eluent switched at 3 min to 75% A and
Preliminary studies at the no carrier added level re-
vealed that the ligands H2PNO, HPN2 and the 5-HT1A
receptor ligand L1 could be labelled with the fac-
[
99mTc(CO)3]+ moiety in good yields (80–85%), being
found for complex 3 an encouraging biological activity
[12]. As part of our ongoing research on the develop-
ment of technetium-labelled receptor-specific agents,
and trying to establish structure biological activity rela-
tionships, we report herein on the derivatization of the
pharmacophore (2-methoxyphenyl)piperazine, part of
the WAY 100635 structure, with phosphinoarylbenzyla-
mide (PO donor atom set) or phosphinoarylbenzylamine
(PN donor atom set) chelator groups, using different
PPh2
O
PPh2
( )n
( )n
N
N
N
N
N
N
H
H
MeO
MeO
L1, n = 1
L2, n = 4
L3, n = 1
L4, n = 4
Scheme 1. Receptor-binding ligands L1–L4.

E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
4813
25% B and at 9 min to 66% A and 34% B followed by a
linear gradient 66% A/34% B to 100% B from 9 to 20
min. The gradient remained at 100% B for 2 min before
switching back to 100% A.
CH2, 4H), 2.32 (t, CH2, 2H), 1.50 (m, CH2, 2H). 31P
NMR (CD3CN): d ꢀ15.4.
L4: The mixture was cooled, and methanol was
added until liberation of H2 stops. The solvents were
evaporated in vacuum to dryness, and the residue was
suspended in MeOH and refluxed for further 30 min.
The solvents were evaporated in vacuum to dryness,
and the residue chromatographed on a silica gel column
with 0.5–4% methanol–chloroform (gradient) to afford a
colorless oil. Yield: 53%, 0.15 g. Anal. Calc. (found) for
C36H44N3OP: C, 76.43 (76.38); H, 7.84 (7.90); N, 7.43
(7.40)%. IR (KBr, m/cmꢀ1): 1495, 1240, 740, 695. 1H
NMR (CDCl3): d 7.47 (m, aromatic, 1H), 7.34–7.21
(m, aromatic, 11H), 7.14 (m, aromatic, 1H), 7.01–6.82
(m, aromatic, 5H), 3.98 (s, CH2, 2H), 3.84 (s, OCH3,
3H), 3.10 (s br, CH2, 4H), 2.65 (s, br, CH2, 4H), 2.48
(t, CH2, 2H), 2.38 (t, CH2, 2H), 1.46 (m, CH2, 2H),
1.36–1.16 (m, CH2, 6H). 31P NMR (CDCl3): d ꢀ15.6.
2.2. Synthesis of N-[4-(6-Aminohexyl)-1-(2-methoxy-
phenyl)piperazine]-2-(diphenylphosphanyl)benzamide
(L2)
N-[2-(Diphenylphosphanyl)benzoyloxy]-succinimide
(500 mg, 1.24 mmol) dissolved in dry dichloromethane
(4 ml) was added dropwise to a stirred solution of 4-
(6-aminohexyl)-1-(2-methoxyphenyl)piperazine (360 mg,
1.24 mmol) in the same solvent (10 ml). After 18 h at
room temperature, the solvent was evaporated to dry-
ness. The obtained residue was chromatographed on
an appropriate column of silica gel with 1–4% metha-
nol/chloroform (gradient) to afford a yellow oil. Yield:
79%, 570 mg. Anal. Calc. (found) for C36H42N3O2P:
C, 74.59 (74.36); H, 7.30 (7.25); N, 7.25 (7.32)%. IR
2.4. General procedure for the preparation of the complexes
fac-[Re(CO3)(j2-L)] (4, L = L2; 5, L = L3; 6, L = L4)
1
(KBr, m/cmꢀ1): 1630 (C@O), 1235, 740, 690. H NMR
(CDCl3): d 7.58 (m, aromatic, 1H), 7.38–7.23 (m, aro-
matic, 12H), 7.01–6.82 (m, aromatic, 5H), 5.92 (t br,
NH, 1H), 3.83 (s, OCH3, 3H), 3.23 (q, CH2, 2H), 3.13
(s br, CH2, 4H), 2.72 (s, br, CH2, 4H), 2.45 (t, CH2,
2H), 1.54 (m, CH2, 2H), 1.36 (m, CH2, 2H), 1.26 (m,
CH2, 4H). 31P NMR (CDCl3): d ꢀ9.7.
A solution of the appropriate ligand L2–L4 (L2: 38
mg, 0.065 mmol; L3 25 mg, 0.048 mmol; L4: 47 mg,
0.083 mmol) in methanol (ca. 2 ml) was added, with stir-
ring, to a solution of (NEt4)2[ReBr3(CO3)] (L2: 50 mg,
0.065 mmol; L3 37 mg, 0.048 mmol; L4: 64 mg, 0.083
mmol) in the same solvent (ca. 3 ml). After refluxing
for 4 h, the reaction mixture was centrifuged to separate
the solid formed in the case of 5, and in the case of com-
plexes 4 and 6, the solvent was evaporated to dryness.
The precipitate (5) or the oily residues (4, 6) were thor-
oughly washed with water and dried under vacuum.
2.3. Synthesis of N-[4-(3-aminopropyl)-1-(2-methoxyphenyl)
piperazine]-2-(diphenyl-hosphanyl)benzylamine (L3) and
N-[4-(6-aminohexyl)-1-(2-methoxyphenyl)piperazine]-
2-(diphenylphosphanyl)benzylamine (L4)
To a solution of L1 (300 mg, 0.57 mmol) or L2 (290
mg, 0.50 mmol) in dry toluene, 1 M borane-dimethylsul-
fide solution in dichloromethane (L1, 1.14 ml, 1.14
mmol; L2, 1.5 ml, 1.50 mmol) was added at room tem-
perature, under a nitrogen atmosphere with stirring. The
solution was allowed to reflux during 24 h under a nitro-
gen atmosphere.
L3: The mixture was cooled, 15 ml of a 10% NaHCO3
solution was added, and refluxed again for 30 minutes.
The solvents were evaporated in vacuum to dryness,
and the residue was treated with 15 ml water and ex-
tracted with dichloromethane (3 · 25 ml). The organic
phases were collected, dried over MgSO4, filtered and
the solvent evaporated. The residue was chromato-
graphed on a silica gel column with 1–3% methanol–
chloroform (gradient) to afford a colorless oil. Yield:
28%, 160 mg. Anal. Calc. (found) for C33H38N3OP Æ -
CH3OH: C, 73.49 (73.83); H, 7.62 (7.99); N, 7.56
(7.59)%. IR (KBr, m/cmꢀ1): 1589, 1500, 1453, 1240,
747, 698 cmꢀ1. 1H NMR (CD3CN): d 7.53 (m, aromatic,
1H), 7.40–7.14 (m, aromatic, 12H), 6.96–6.84 (m, aro-
matic, 5H), 3.94 (s, CH2, 2H), 3.77 (s, OCH3, 3H),
2.92 (s br, CH2, 4H), 2.55 (t, CH2, 2H), 2.47 (s, br,
2.5. fac-[Re(CO3)(k2-L2)] (4)
The complex, which was obtained as an yellow oil,
could be recrystallized from CH2Cl2/Hexane giving
pale-yellow crystals suitable for X-ray diffraction analy-
sis. Yield: 38 mg, 69%. Anal. Calc. (found) for
C39H42N3O5PRe Æ HBr: C, 46.34 (46.03); H, 4.29
(4.35); N, 4.16 (4.32)%. IR (KBr, m/cmꢀ1): 2010, 1910,
1870, 1590 (C@O), 1235, 740 and 690 cmꢀ1. H NMR
1
(CDCl3): d 7.68–7.55 (m, aromatic, 4H), 7.46–7.43 (m,
aromatic, 6H), d (7,37–7.26, m, aromatic, 3H), 7.17–
6.78 (m, aromatic, 5H), 3.86 (s, OCH3, 3H), 3.58–3.47
(m, CH2, 6H), 3.18–3.04 (m br, CH2, 2H), 1.86 (s br,
CH2, 4H), 1.62 (s br., CH2, 8H). 31P NMR (CDCl3): d
14.9.
2.6. fac-[Re(CO3)(j2-L3)] (5)
The complex was obtained in the form of a white so-
lid, which could be recrystallized from CH2Cl2/hexane
yielding pale-yellow crystals suitable for X-ray diffrac-
tion analysis. Yield: 19 mg, 46%. Anal. Calc. (found)

4814
E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
Table 1
Crystal data for [Re(CO3)Br(j2-L2)] (4) and [Re(CO3)Br(j2-L3)] (5)
for C36H38N3O4PBrRe Æ H2O: C, 48.48 (48.53); H, 4.52
(4.48); N, 4.71 (4.96)%. IR (KBr, m/cmꢀ1): 2010, 1925,
1
1890, 1260, 800 cmꢀ1. H NMR (CDCl3): d 7.54–7.28
Complex
4
5
(m, aromatic, 13H), 6.95 (t, aromatic, 2H), 6.88–6.79
(m, aromatic, 3H), 4.28 (d, CH, 1H, J = 12 Hz), 4.05
(d, CH, 1H, J = 11 Hz), 3.82 (s, OCH3, 3H), 3.30 (s
br, CH2, 3H), 3.12 (s br, CH2, 2H), 2.86–2.65 (m,
CH2, 7H), 2.05 (s br, CH, 1H), 1.58 (s br, CH, 1H).
31P NMR (CDCl3): d 11.7.
Empirical formula
Crystal size, mm3
Formula weight
Crystal system
Space group
C39H42BrN3O5PRe C36H38BrN3O4PRe
0.55 · 0.16 · 0.16
929.84
Triclinic
0.76 · 0.54 · 0.36
873.77
Triclinic
ꢀ
ꢀ
P1
P1
˚
a (A)
8.8133(14)
15.893(4)
15.968(4)
61.225(19)
74.324(16)
84.612(18)
1885.8(8)
2
11.238(3)
11.656(3)
14.4143(15)
91.818(15)
92.986(11)
112.03(2)
1745.2(6)
2
˚
b (A)
˚
c (A)
2.7. fac-[Re(CO3)(k2-L4)] (6)
a (ꢁ)
b (ꢁ)
c (ꢁ)
3
˚
The complex was obtained as an yellow oil. Yield: 52
mg, 68%. Anal. Calc. (found) for C39H44N3O4PBrRe: C,
51.15 (51.22); H, 4.84 (4.79); N 4.59 (4.55)%. IR (KBr, m/
cmꢀ1): 2020, 1920, 1870, 1245, 750, 695. 1H NMR
(CDCl3): 7.55–7.31 (m, aromatic, 13H), 7.05 (m, aro-
matic, 1H), 6.94–6.76 (m, aromatic, 4H), 4.21 (d, CH,
1H, J = 12 Hz), 4.08 (d, CH, 1H, J = 12 Hz), 3.84 (s,
OCH3, 3H), 3.49 (m, CH2, 6H), 3.32 (m, CH2, 2H),
3.09–2.90 (m, CH2, 6H), 2.00 (s br, CH2, 2H), 1.77 (s
br, CH2, 2H), 1.44 (m, CH2, 2H). 31P NMR (CDCl3):
d 10.7.
Volume (A )
Z
qcalc. (g cmꢀ3
)
1.638
4.370
1.663
4.714
l(Mo Ka) (mmꢀ1
No. reflections measured 7047
)
7349
6575 [Rint = 0.0352] 6939 [Rint = 0.0493]
No. unique reflections
a
R1
wR2
0.0846
0.1920
0.0470
0.1031
a
P
P
P
2
a
R1 ¼
jj F o j ꢀ j F c jj = j F o j and wR2 ¼ ½ ðwðF 2o ꢀ F c2Þ Þ=
P
ðwðF 2oÞ Þꢁ1=2. The values were calculated for data with I > 2r(I).
2
(L1–L4) in EtOH was added. The vial was then heated
(L1, 100 ꢁC/30 min; L2 100 ꢁC/60 min; L3 100 ꢁC/30
min; L4 100 ꢁC/60 min), and the final solution ana-
lysed by HPLC.
2.8. X-ray crystallographic analysis
Pale-yellow crystals of 4 and 5, suitable for X-ray dif-
fraction analysis, were obtained from a mixture of
CH2Cl2/hexane and were fixed inside thin-walled glass
capillaries. Data were collected at room temperature
on an Enraf–Nonius CAD4-diffractometer with graph-
ite-monochromatized Mo Ka radiation, using a ꢀ2h
scan mode. Unit cell dimensions were obtained by
least-squares refinement of the setting angles of 25
reflections with 14.0 < 2h < 28.0ꢁ for 4 and 16.8 <
2h < 27.9ꢁ for 5. A summary of the crystallographic data
is given in Table 1. Data were corrected for Lorentz and
polarization effects and for absorption by empirical cor-
rections based on W scans [20]. The heavy atom posi-
tions were located by Patterson methods using
SHELXS-97 [21]. The remaining atoms were located by
sucessive difference Fourier techniques and refined by
least-squares refinements on F2 using SHELXL-97 [22].
All the non-hydrogen atoms were refined with aniso-
tropic thermal motion parameters and the contribution
of the hydrogen atoms were included in calculated posi-
tions. Atomic scattering factors and anomalous disper-
sion terms were taken from [20]. The drawings were
made with ORTEP-3 [23].
2.10. Receptor biding assays on rat brain homogenates
The 5HT1A receptor binding assays were performed
using [3H]-8-OH-DPAT as radioligand and rat hippo-
campus homogenate. The binding assay was carried
out in a final volume of 2.5 ml Tris–HCl buffer (50
mM, pH 7.4, 0.1% ascorbic acid, 2 mM CaCl2) con-
taining 0.10 nM [3H]-8-OH-DPAT, membran homo-
genate (about 20 lg/ml protein), and various
concentration of the rhenium complexes 3–6. The com-
plexes were dissolved in DMSO up to 1 nM, then di-
luted with buffer. Non-specific binding was defined as
the amount of [3H]-8-OH-DPAT bound in the presence
of serotonin (Sigma). The samples were incubated in
triplicates at 20ꢁC for 120 min. The incubation was
terminated by rapid filtration through GF/B glass fiber
filters (Whatman) using a 30-port Brandel Cell Har-
vester. The filters were rapidly washed with four 4 ml
portions of ice-cold buffer, transferred into 10 ml scin-
tillation fluid (Ultima-Gold, Packard) and analysed for
radioactivity.
2.9. Synthesis at tracer level, 99mTc-complexes (3a–6a) –
general method
The 5HT2A receptor binding assays: the cortex of rat
brain was homogenized in ice-cold buffer (50 mM Tris–
HCl, pH 7.6) with an Ultra-Turrax T 25. The homogen-
ate was centrifuged at 20,000g for 10 min. The resulting
pellet was resuspended and centrifuged under above
conditions. After repeating the same procedure the
To a solution of the organometallic precursor fac-
99mTc(OH2)3(CO)3]+ (900 ll; 3–4 mCi) was added
[
100 ll of a 10ꢀ3 M stock solution of the ligands

E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
4815
pellet was resuspended in 10 ml of buffer and stored at
ꢀ20 ꢁC.
[3H]ketanserin (from NEN) was used as a radiolig-
O
PPh2
O
( )n
H2N
N
N
O
N
+
and. The binding assay was carried out in a final volume
of 5 ml Tris–HCl buffer, pH 7.6, containing 0.12 nM
[3H]ketanserin, membrane homogenate (about 20 lg/ml
protein), and various concentrations of the Re com-
plexes. The complexes were dissolved in DMSO up to
1 nM, then diluted with buffer. Non-specific binding
was defined as the amount of [3H]ketanserin bound in
the presence of 1 lM mianserin (Sigma). The samples
were incubated in triplicates at 20 ꢁC for 60 min. Incuba-
tion, filtration and counting of the samples were the
same as described above.
MeO
O
(n = 1, 4)
i
PPh2
O
( )n
N
N
N
H
MeO
(n = 1, L1; n = 4, L2)
ii
PPh2
3. Results and discussion
( )n
N
N
N
H
The bifunctional chelating agents (BFCA) L1 and L2
have been prepared, similarly to a previously published
method, by reaction of N-[2-(diphenylphosphanyl)-
benzoyloxy]succinimide with 4-(3-aminopropyl)-1-(2-
methoxyphenyl)-piperazine and 4-(6-aminohexyl)-1-(2-
methoxyphenyl)piperazine, respectively [11,12]. The
BFCA L3 and L4 were obtained by reduction of the
amide groups in L1 and L2 with BH3 Æ SMe2 in refluxing
toluene, respectively (Scheme 2).
The compounds L2–L4 were characterized by 31P and
1H NMR spectroscopy, elemental analysis and IR
spectroscopy. In the IR spectra of L3 and L4 no stretch-
ing bands in the range 1630–1640 cmꢀ1 were found, con-
firming the absence of the carbonyl amide function. As
expected, the 31P NMR spectra show only one signal
for the phosphorus atom for all compounds (L1,
d ꢀ8,3; L2, d ꢀ9.7; L3, d ꢀ5.4; L4, d ꢀ15.6). The rhe-
nium tricarbonyl complexes fac-[Re(CO)3Br- (j2-L)]
(3, L = L1; 4, L = L2; 5, L = L3; 6, L = L4), were
obtained by reacting L1–L4 with the precursor
(NEt4)2[ReBr3(CO)3] under refluxing methanol, as de-
picted in Scheme 3. These compounds have been used
as surrogates for 99mTc-complexes (3a–6a).
MeO
(n = 1, L3; n = 4, L4)
Scheme 2. Synthesis of the ligands L1–L4. (i) Dichloromethane, room
temperature; (ii) BH3 Æ SMe3, toluene, reflux.
MeO
N
N
( )n
L1, L2
H
N
O
Re
Br
Ph
Ph
P
OC
CO
CO
2-
Br
Br
Br
(n = 1, 3
n = 4, 4)
(i)
Re
CO
OC
CO
MeO
N
While fac-[Re(CO)3Br(j2-L1)] (3) and fac-[Re-
(CO)3Br(j2-L3)] (5) were isolated as white solids, which
precipitate during reaction course or work-up, com-
plexes fac-[Re(CO)3Br(j2-L2)] (4) and fac-[Re(CO)3Br-
(j2-L4)] (6) were obtained as viscous colorless oils after
evaporation of the solvent and washing of the residues
with water. The neutral complexes 3–6 are air and mois-
ture stable, being soluble in halogenated solvents. All the
complexes have been fully characterized by 1H/31P NMR
and IR spectroscopy and, in the cases of 4 and 5, by X-
ray diffraction analysis. The L2 receptor-binding ligand
in complex 4 coordinates as neutral and bidentate
through the phosphorus and the oxygen atom of the car-
bonyl, in a similar way to what has been previously ob-
served for L1 in complex 3 [11]. The receptor-binding
N
( )n
L3, L4
H
N
Ph
Ph
P
Br
Re
OC
CO
CO
(n = 1, 5
n = 4, 6)
Scheme 3. Synthesis of the complexes 3–6. (i) Methanol, reflux.
ligands L3 and L4 coordinate also as neutral and biden-
tate, through the phosphorus and the nitrogen atom of
the secondary amine. IR spectra were consistent with
the fac-[M(CO)3] geometry of compounds 4–6, as

4816
E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
previously observed for complex 3 [11]. Very strong
m(CO) stretching bands appear at 2010, 1910 and 1870
cmꢀ1 for 4; 2010, 1925 and 1890 cmꢀ1 for 5; 2020, 1920
and 1870 cmꢀ1 for 6. The m(C@O) stretching vibration
of the carbonyl group of the receptor-binding ligand
L2 in complex 4 appears at 1590 cmꢀ1, i.e. 40 cmꢀ1 lower
in energy relatively to the corresponding free ligand
(1630 cmꢀ1), which is a strong evidence for metal coordi-
nation through the carbonyl function. Complexes 4–6
present only one signal in the 31P NMR spectra (4, d
14.9; 5, d 11.7; 6, d 10.7 ppm), strongly downfield shifted
relatively to the corresponding free ligands (L2, d ꢀ9.7;
L3, d ꢀ15.4; L4, d ꢀ15.6 ppm), confirming a strong r-
donor character for the phosphorus atom.
clear units. The ORTEP views of the molecular
structures of 4 and 5 are given in Figs. 1 and 2, respec-
tively. The bond distances and angles are unexceptional,
as shown in Tables 3 and 4. The Re atom in the neutral
complexes 4–5 is six-coordinated, exhibiting a slightly
distorted octahedral coordination geometry. The three
carbonyl ligands occupy one triangular face of the coor-
dination polyhedron, being the other three remaining
coordination sites defined by a bromide and by the
bidentate L2 and L3 ligands in 4 and in 5, respectively.
Deviations from the idealized octahedral geometry
can be seen on the bond angles around the Re atom
(Tables 3 and 4). The cis and trans bond angles range
between 80.4(3)–99.7(6)ꢁ and 169.8(5)–179.5(6)ꢁ, and
between 82.84(15)–94.3(3)ꢁ and 174.2(2)–177.1(3)ꢁ in 4
and 5, respectively. In these complexes the bidentate che-
lators form non-planar six-membered rings which pre-
sent distorted half-boat and boat conformations in 4
([Re, O4,C4, C111, C110, P1]) and 5 ([Re, N1, C4,
C111, C110, P1]), respectively. All the above referred
parameters indicate for 4 an higher distortion from the
octahedral geometry than for 5, which can certainly be
due to the sp2 hybridization of the C4 atom in complex 4.
In 4 and 5 the metrical parameters are comparable to
those that we have previously reported for other rhe-
nium tricarbonyl complexes also anchored by hetero-
functionalized phosphines [11,12]. The average Re–CO
1
The most important feature of the H NMR spectra
of complexes 5 and 6 is related with the diastereotopic
character of the two methylenic protons near to the phe-
nyl ring in L3 and L4 upon coordination to the metallic
centre (5: d 4.28, d, J = 12 Hz, 1H; d 4.05, d, J = 11 Hz,
1H; 6: d 4.21, d, J = 12 Hz, 1H; d 4.08, 1H, d, J = 12
Hz).
We have evaluated the possibility of preparing the
complexes 3–6 at the no carrier added level, using fac-
[
99mTc(OH2)3(CO)3]+ as precursor. Thus, the neutral
99mTc(I) tricarbonyl complexes, analogues of the Re
complexes with the receptor-binding ligands L1–L4,
[
99mTc(CO)3Cl(j2-L1)] (3a),
[
99mTc(CO)3Cl(j2-L2)]
(4a), [99mTc(CO)3Cl(j2-L3)] (5a), and [99mTc(CO)3Cl
(j2-L4)] (6a) have been prepared. These complexes have
been obtained in relatively high yields (87–98%), using a
10ꢀ4 M final ligand concentration (Table 2).
bond distances in 4 (average 1.92(2) A) and 5 (average
˚
˚
1.93(1) A) are similar and in the range found for other
neutral Re(I) tricarbonyl compounds containing
monodentate, bidentate or tridentate ligands (1.89–
The characterization of 3a–6a was made by compar-
ing their radioactive traces on the HPLC with the UV
traces (monitored at 254 nm) of the analytically pure
Re analogues (Table 2).
N1
4. Description of the structures
O4
The structures of complexes [Re(CO)3Br(j2-L2)] (4)
and [Re(CO)3Br(j2-L3)] (5) consist of discrete mononu-
N2
Br1
C2
Re
P1
O2
Table 2
Labelling conditions of ligands L1–L4; HPLC retention times (Rt) of
complexes 3a–6a (99mTc) and of the analogues Re compounds 3–6
C3
O3
N3
C1
O1
Ligands Labelling conditions
Yields (%)a
Reaction time (min) Rt (min)
Reb
99mTcc
O5
L1
L2
L3
L4
30
60
30
60
3 – 22.7
4 – 21.5
5 – 21.7
3a – 22.5 3a – 95
4a – 23.3 4a – 98
5a – 22.7 5a – 87
6 – 22.05 6a – 23.6 6a – 92
a
b
c
Addition of 1% SDS after labelling; [L] = 10ꢀ4 M, 100 ꢁC.
UV traces monitored at 254 nm.
Radioactive traces.
Fig. 1. ORTEP view of the neutral complex fac-[Re(CO)3Br(j2-L2)]
(4). Vibrational ellipsoids are drawn at the 20% probability level.

E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
4817
Table 4
Selected bond lengths (A) and angles (ꢁ) for [Re(CO3)Br(j -L3)] (5)
2
˚
Re–C(1)
Re–C(2)
Re–C(3)
1.899(10)
1.940(10)
1.951(9)
Re–N(1)
Re–P(1)
Re–Br(1)
2.237(6)
2.4703(19)
2.6118(11)
O4
C(1)–Re–C(2)
C(1)–Re–C(3)
C(2)–Re–C(3)
C(1)–Re–N(1)
C(2)–Re–N(1)
C(3)–Re–N(1)
C(1)–Re–P(1)
C(2)–Re–P(1)
89.3(4)
90.5(4)
90.6(3)
177.1(3)
93.5(3)
88.8(3)
91.7(3)
92.4(2)
C(3)–Re–P(1)
N(1)–Re–P(1)
C(1)–Re–Br(1)
C(2)–Re–Br(1)
C(3)–Re–Br(1)
N(1)–Re–Br(1)
P(1)–Re–Br(1)
176.3(2)
88.94(15)
94.3(3)
N2
174.2(2)
84.9(3)
N3
82.84(15)
92.00(5)
N1
Br
In complex 5 an intramolecular hydrogen bridge be-
˚
O3
O2
C3
tween N1 and N2 was observed (N1ꢂ ꢂ ꢂN2, 2.932 A;
N1–Hꢂ ꢂ ꢂN2, 144.01ꢁ), forcing a folding of the pharma-
cophore towards the metal center.
Re
P1
C2
4.1. Receptor binding assays on rat brain homogenates
C1
The rhenium (I) tricarbonyl complexes 3–6 have been
used for determination of the receptor-binding affinity
and selectivity in rat brain homogenates. The IC50 val-
ues of these compounds for 5-HT1A and 5-HT2A recept-
ores, summarized in Table 5, were determined by
displacement studies.
O1
From the values found we can conclude that complex
3 is the only one which presents a relatively interesting
affinity and specificity toward the 5HT1A receptors for
further biological evaluation (5HT1A, IC50: 20 nM;
5HT2A, IC50: 4680 nM).
Fig. 2. ORTEP view of the neutral complex fac-[Re(CO)3Br(j2-L3)]
(5). Vibrational ellipsoids are drawn at the 30% probability level.
For all the other complexes the affinity and specificity
towards 5HT1A decrease significantly. The fit of the mol-
ecule into the binding pocket of the receptors was signif-
icantly affected when an amide group in the chelate unit
(3, 5HT1A, IC50: 20 nM) was replaced by a secondary
amine (5, IC50: 5HT1A, 285 nM). This effect is even more
pronounced when the alkyl chain length increases (3 ver-
sus 4 and 5 versus 6).
Taking into account previous results on structure/ac-
tivity relationships and also considering that what is ra-
tional and predictable in the design of organic receptor
ligands cannot simply be extended to technetium com-
pounds, we think that the somehow encouraging results
Table 3
˚
Selected bond lengths (A) and angles for (ꢁ) for complex
[Re(CO3)Br(j2-L2)] (4)
Re–C(1)
Re–C(2)
Re–C(3)
1.913(17)
1.914(18)
1.94(2)
Re–Br
Re–O(4)
Re–P
2.6382(19)
2.162(10)
2.444(4)
C(1)–Re–C(2)
C(1)–Re–C(3)
C(2)–Re–C(3)
C(1)–Re–O(4)
C(2)–Re–O(4)
C(3)–Re–O(4)
C(1)–Re–P(1)
C(2)–Re–P(1)
90.5(8)
89.5(7)
87.8(7)
179.5(6)
89.4(6)
91.0(5)
99.7(6)
169.8(5)
C(3)–Re–P(1)
O(4)–Re–P(1)
C(1)–Re–Br(1)
C(2)–Re–Br(1)
C(3)–Re–Br(1)
O(4)–Re–Br(1)
P(1)–Re–Br(1)
92.2(5)
80.4(3)
92.0(5)
90.9(5)
178.0(5)
87.5(3)
88.82(11)
Table 5
Inhibition constants (IC50) of 3–6 for 5HT1A and 5HT2A receptors
˚
2.03 A) [11]. Likewise, the Re–P bond distances found in
˚
˚
4 (2.444(4) A) and 5 (2.470(2) A) and the Re–Br bond
Complex
IC50 (nM)
˚
˚
lengths are normal (4, 2.6382(19) A; 5, 2.6118(11) A),
comparing well with the values described previously
for other tricarbonyl rhenium complexes [11]. The Re–
5-HT1A
[3H]–OH–DPAT
5-HT2A
[3H]ketanserin
5-HT2A/5-HT1A
3
4
5
6
20 0.1 nM
200 4 nM
285 4 nM
1100 4 nM
4680 0.1 nM
340 9 nM
490 8 nM
234
1.7
1.7
1.0
˚
N bond distance of 2.237(6) A in 5 is typical of a single
bond and agrees with the presence of an sp3 hybridized
amine nitrogen [24].
1190 0.5 nM

4818
E. Palma et al. / Journal of Organometallic Chemistry 689 (2004) 4811–4819
obtained for compound 3 can be due to a certain rigidity
imposed by the presence of the amide group, which de-
creases when the coordination to the metal is done
through a secondary amine. On contrary, increasing
the alkyl chain length a higher mobility of the pendant
arm is observed with a folding towards the metal center.
In the case of compound 5 this folder seems to be fa-
voured due to an intramolecular hydrogen bridge be-
tween the secondary amine of the piperazine (Figs. 1
and 2). Whether this type of interactions are maintained
in solution is difficult to say, although the mobility of
the pendant arm seems to exist in solution as indicated
by the broadness of the piperazine resonances observed
Cambridge CB2 1EZ, UK; Tel.: +44-1223-336408; fax:
+44-1223-336003; e-mail: deposit@cdcc.cam.ac.uk;
www: http://ccdc.cam.ac.uk
Acknowledgement
This work was partially supported by the POCTI/
QUI/3542/2000 and by COST ACTION B12. Elisa Pal-
ˆ
ma thanks the Fundac¸a˜o para a Ciencia e Tecnologia
for a BIC Grant.
1
in the H NMR spectra of 5.
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