Preparation, characterization, and screening of a high affinity organometallic probe for α-adrenergic receptors

Article abstract of DOI:10.1021/jm2001162

Figure Presented. An organometallic rhenium complex having high and selective affinity for α-adrenergic receptors is reported. A series of methoxyphenylpiperazinecarborane ligands complexed to the [Re(CO) ₃]⁺ core were prepared and t

Full text of DOI:10.1021/jm2001162

ARTICLE
pubs.acs.org/jmc
Preparation, Characterization, and Screening of a High Affinity
Organometallic Probe for r-Adrenergic Receptors
Anika S. Louie,† Neil Vasdev,‡ and John F. Valliant*,†,§
†Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street W., Hamilton, Ontario, L8S 4M1, Canada
‡Centre for Addiction and Mental Health and Department of Psychiatry, University of Toronto, 250 College Street, Toronto, Ontario,
M5T 1R8, Canada
§Department of Medical Physics and Applied Radiation Sciences, McMaster University, 1280 Main Street W., Hamilton, Ontario,
L8S 4M1, Canada
S Supporting Information
b
ABSTRACT: An organometallic rhenium complex having high
and selective affinity for R-adrenergic receptors is reported. A
series of methoxyphenylpiperazinecarborane ligands com-
plexed to the [Re(CO)3]þ core were prepared and the resulting
organometallic compounds screened for binding to serotonin,
σ, and adrenergic receptors as well as dopamine, serotonin, and
norepinephrine transporters. An amide linked derivative, for which a single crystal X-ray structure was obtained, showed high and
selective affinity toward R1A, R1D, and R2C adrenergic receptors with Ki values of 17, 21, and 39 nM, respectively. A method to
prepare the corresponding 99mTc complex was also developed where the resulting metallocarborane was shown to be stable and is
therefore suitable for in vivo molecular imaging studies.
’ INTRODUCTION
Carboranes are attractive ligands for bioorganometallic chem-
istry because they are stable in water, the cluster can be labeled
with a range of different radionuclides, and they can be readily
derivatized using a number of different strategies. These char-
acteristics provide advantages over Cp which is challenging to
radiolabel and derivatize because it frequently undergoes un-
wanted side reactions including dimerization.9ꢀ11 These issues
complicate the creation of libraries of candidates and develop-
ment of adequate structureꢀactivity relationship (SAR) studies
when searching for new MIPs. Building on the advantages offered
by carboranes, a family of novel carborane-1-(2-methoxyphenyl)
piperazine (WAY) conjugates was synthesized and the corre-
sponding rhenium complexes were isolated and screened
against a panel of CNS targets. The compounds are designed
to be used to image targets located in the brain if the anionic
metal complexes can cross the bloodꢀbrain barrier, or alter-
natively they will be used for imaging targets in the periphery
if they cannot. For the highest affinity compound a method
to produce the radioactive analogue (99mTc) was also
developed.
There has been growing interest in using organometallic
chemistry to create high affinity molecular imaging probes
(MIPs).1ꢀ5 While most bioorganometallic probes are derived
from cyclopentadiene (Cp), the strategy employed here utilized
metallocarboranes which resulted in the discovery of the first
organometallic MIP having high affinity for R-adrenergic
receptors.
MIPs can be used to noninvasively monitor changes in protein
expression associated with disease onset and progression, to
determine optimal dosing levels and schedules, and to predict
how patients and preclinical models will respond to therapies.6 In
central nervous system (CNS) imaging, for example, the appro-
priate MIP can be used with positron emission tomography
(PET) or single photon emission computed tomography (SPECT)
to identify changes in levels of active neuroreceptors and to
visualize accumulation of β-amyloid or microglia as a means to
help with the diagnosis of neurological diseases or disorders.
Gmeiner and co-workers demonstrated that it is possible to
prepare high affinity organometallic probes for key proteins in
the CNS. In their study, an arylpiperazine was linked to ferrocene
where the complex showed exceptionally high binding to both
the dopamine D4 receptors (Ki = 0.52 nM) and serotonin
5-HT1A (Ki = 0.50 nM) (Figure 1a).7 When Alberto et al. linked
the same vector to CpRe(CO)3 (Figure 1b) through a different
’ RESULTS AND DISCUSSION
A series of arylpiperazinecarborane derivatives were prepared
having different tether lengths and linkages between the cluster
and the targeting vector. These variations have been shown to
result in significant changes in binding affinities and target selectivity
spacer group, the compound exhibited high affinity (IC50
=
6 nM) for 5-HT1A.8 Beyond these Cp-derived compounds, there
are to our knowledge no other high affinity organometallic
probes for the CNS that have been reported to date.
Received: February 1, 2011
Published: March 23, 2011
r
2011 American Chemical Society
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profiles.12 Attempts to convert 1-(2-methoxyphenyl)-4-(2-propy-
nyl)piperazine (1) and 1-(2-methoxyphenyl)-4-(4-pentynyl)piper-
azine (2), which were prepared by simple alkylation with the
appropriate haloalkyne,13 to the corresponding clusters were un-
successful using conventional methods. Success in isolating 3 and 4
was achieved using decaborane (B10H14) and a catalytic amount of
ionic liquid (1-butyl-3-methylimidazolium chloride) in boiling
toluene (Scheme 1a).14,15
Preparation of the amide linked cluster 5, which can be considered
a carborane analogue of the compound reported by Gmeiner and co-
workers,7 was achieved by combining the acid chloride 6with amine 7
(Scheme 1b). Forceful reaction conditions were needed, which is
likely due to the proximity of the acid to the extremely bulky cluster.
Coupling also resulted in the formation of the nido-carborane with the
protonated salt of the excess amine as the counterion. To ensure that
the counterion would not interfere with the in vitro binding assays, a
simple salt exchange using sodium hydroxide was performed.
The preparation of the nido-forms of 3 and 4 was accom-
plished using sodium fluoride16 immediately prior to complexa-
tion with the [Re(CO)3]þ core. Repeated microwave heating in
the presence of excess [Re(CO)3(H2O)3]Br was used to gen-
erate the rhenacarborane complexes 8, 9, and 10 in 82%, 95%,
and 10% yields, respectively (Scheme 1c).17 The rhenium
complexes were isolated by column chromatography where
LCꢀMS showed a single peak in the UV spectrum and the
expected mass and isotope pattern in the mass spectrum, which
1
for ReꢀB9 clusters is distinct. H NOE experiments indicated
that compounds 8 and 10 were 2, 1, 8 clusters while compound
Figure 1. Organometallic derivatives of 1-(2-methoxyphenyl)piperazine
(WAY): (a) WAY ferrocenylcarboxamide7 and (b) WAY CpRe(CO)3
derivatives.8
Figure 2. Thermal ellipsoid plot of 10 (50% probability ellipsoids).
Hydrogen atoms have been omitted for clarity.
Scheme 1. Preparation of Compounds 3ꢀ13 (Solid Black Spheres = BH)a
a Full structures for all compounds can be found in the Supporting Information.
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Table 1. Binding Affinities of Rhenacarborane Complexes 8ꢀ10 to Serotonin, Adrenergic and σ Receptors
Ki (nM)
serotonin
5-HT2B
adrenergic
R1D
σ
compd
5-HT1A
5-HT1D
5-HT6
5-HT7
R1A
R1B
R2C
σ1
σ2
8
834 ( 54
118 ( 8
a
>1000
>1000
>1000
>1000
692 ( 77
a
a
a
>1000
a
270 ( 15
174 ( 9
21 ( 1
575 ( 25
244 ( 10
39 ( 2
a
a
a
a
>1000
>1000
201 ( 9
a
9
>1000
211 ( 16
128 ( 7
302 ( 11
115 ( 6
17 ( 1
62 ( 4
122 ( 7
435 ( 39
162 ( 9
10
>1000
40 ( 5
WAY100635
0.5 ( 0.04
661 ( 82
248 ( 23
36 ( 1
562 ( 28
a In the primary binding assay <50% inhibition of specific binding was measured.
9 remained 3, 1, 2. These configurations are consistent with our
previous reports where nido-carboranes isomerize upon com-
plexation with Re(I) when sterically demanding or electron
withdrawing groups are located immediately adjacent to the
cluster.18 An X-ray quality crystal of 10 (Figure 2) was obtained
from slow evaporation of methanol, confirming that the isomer is
in fact 2, 1, 8 which is consistent with the NMR data.
Given the positive screening results, a method to prepare the
technetium-99m analogue of 10 was developed. nido-Carborane
5 as the sodium salt was combined with [99mTc(CO)3(H2O)3]þ,
which was prepared using a standard kit method (Scheme 1c).
After microwave heating for 15 min at 195 °C, the desired
product formed as the major radioactive product. HPLC pur-
ification, which is not absolutely necessary given the formation of
a single radiolabeled compound, can be used to remove residual
ligand but it reduces the radiochemical yield (14%). By use of
either approach, sufficient quantities of 13 can be prepared for
preclinical imaging studies. Analytical HPLC analysis showed
that the retention time of the radioactive product matched that
for the analogous rhenacarborane complex (observed using
UVꢀHPLC). For comparison, the nido-carboranes of 3 and 4
were also labeled under similar conditions and the radiochemical
yields were comparable. The stabilities of the 99mTc complexes
(11ꢀ13) were tested by resuspending the products in ethanol/
saline and checking the purity by HPLC under different elution
solvents and gradients. There were no signs of degradation in any
case for up to 6 h, which is consistent with our previous stability
data on radiometallocarborane complexes.24
R-Adrenergic receptors are of particular interest for molecular
imaging applications because of their involvement in contraction
and growth of smooth and cardiac muscle, regulation of arterial
blood pressure,25 cognitive function such as attention and memory,26
and motor activity.27 Studies have also indicated the involvement
of R-adrenergic receptors in pain28 and Alzheimer’s disease26
through abnormalities in neurotransmission. The exact role of
this system in normal and disease physiology has been difficult
to study because of a lack of a suitable molecular imaging probe.29
While a high affinity metallocarborane for the R-class of adre-
noceptors is a major step toward addressing this issue, the
carborane complexes have a negative charge which will likely
render these probes useful for studying peripheral R-adrenergic
receptors only. We are therefore currently using 13 as a platform
for making neutral analogues to facilitate crossing of the bloodꢀ
brain barrier and as the basis for identifying even higher affinity
analogues by exploiting the versatile chemistry of carboranes.30,31
To assess the affinity and selectivity of the metallocarboranes
for several CNS targets, a series of in vitro binding assays were
performed. All screens were run in duplicate and in parallel with
the 5-HT1A receptor antagonist N-{2-[4-(2-methoxyphenyl)-
1-piperazinyl] ethyl}-N-(2-pyridyl)cyclohexanecarboxamide
(WAY100635) as a positive control which showed affinities that
are consistent with literature values for 5-HT1A binding.19
Compounds 8ꢀ10 were screened for binding to both serotonin
(5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C,
5-HT3, 5-HT5A, 5-HT6, 5-HT7) and adrenergic receptors (R1A,
R1B, R1D, R2A, R2B R2C, β1, β2, and β3), as well as dopamine,
serotonin, and norepinephrine transporters (DAT, SERT, and
NET, respectively) and σ1 and σ2 receptors; findings are
summarized in Table 1. The complete binding assay results of
all the receptors are available in the Supporting Information.
Derivatives 8ꢀ10 showed either less than 50% of specific binding
in the primary assay or >1000 nM binding for 5-HT1B, 5-HT1D
,
5-HT1E, 5-HT2A, 5-HT2C, 5-HT3, and 5-HT5A serotonergic
receptors, adrenergic receptors (R2A, R2B, β1, β2, and β3), σ1-
receptor, and DAT, NET, and SERT transporters.
Moderate to poor binding was observed for complexes 8ꢀ10
for the serotonin 5-HT1A, 5-HT2B, and 5-HT6 receptors and for
the R1B adrenergic receptor. Interestingly, the amide linked
cluster 10 showed high affinity toward 5-HT7 serotonergic
receptor (40 nM), adrenoceptors R1A (17 nM), R1D (21 nM),
and R2C (39 nM) with modest affinity for the σ2 receptor (201
nM). While there are a number of reported PET probes derived
primarily from carbon-11 or fluorine-18 for imaging adrenergic
receptors20,21 and SPECT probes using 99mTc,22 there are no
examples of organometallic agents which in the case of techne-
tium is extremely desirable because of the isotope’s widespread
use in nuclear medicine and low cost.
Compound 10 is a unique example of a technetium probe with
high affinity for three R-adrenergic subtypes (1A, 1D, and 2C). An
additional attractive feature of these compounds is the low to
negligible affinity toward σ receptors, which are abundant in several
CNS regions and can therefore affect target to nontarget ratios. The
selectivity of the compound must be influenced by the cluster, since
arylpiperazine derivatives have often been shown to bind to both
serotonin and adrenergic receptors, which is due to the high degree
of homology between the receptors (approximately 45%).23
’ EXPERIMENTAL SECTION
Reagents and General Procedures. Unless otherwise stated, all
chemicals and reagents were purchased and used as received from
Sigma-Aldrich without further purification. Decaborane and o-carborane
were obtained and used as received from Katchem Ltd., and 1-butyl-
3-methylimidazolium chloride ([BMIM][Cl]) was purchased from
Strem Chemicals. Compounds prepared according to literature pro-
cedures were 1-(2-methoxyphenyl)-4-(prop-2-ynyl)piperazine (1),13
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1,2-dicarba-closo-dodecaborane-1-carboxylic acid,32 [Re(CO)3
(H2O)3][Br],33 and [99mTc(CO)3(H2O)3]þ.34
(solvent A = acetonitrile (0.1% formic acid), solvent B = water (0.1%
formic acid), 1 mL/min) 70:30 A/B isocratic for 15 min; method E
(solvent A = acetonitrile (0.1% formic acid), solvent B = 20 mM
ammonium acetate, 1 mL/min) 70:30 A/B isocratic for 15 min; method
F (solvent A = acetonitrile, solvent B = 0.1 M ammonium formate,
1 mL/min) 70:30 A/B isocratic for 15 min; method G (solvent A =
methanol, solvent B = water, 1 mL/min) 70:30 A/B isocratic for 15 min.
1-(2-Methoxyphenyl)-4-(pent-4-ynyl)piperazine (2). The
synthesis was adapted from a procedure reported by Khatuya and co-
workers.13 Sodium iodide (1.2 g, 8.0 mmol) was added to a solution of
5-chloro-1-pentyne (0.83 mL, 7.8 mmol) in acetonitrile (20 mL), and
the resulting mixture was stirred at 25 °C for 1 h. A solution of 1-(2-
methoxyphenyl)piperazine (1.0 g, 5.2 mmol) in acetonitrile (15 mL)
was added dropwise to the reaction mixture followed by a single addition
of potassium carbonate (9.4 g, 68 mmol). The mixture was heated at
reflux (82 °C) for 48 h. The mixture was cooled to room temperature,
and distilled water (50 mL) was added. The product was extracted with
ethyl acetate (3 ꢁ 30 mL); the combined organic fractions were dried
over anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure. The product was purified by either flash or automated
(40þM) silica column chromatography using a gradient of 6ꢀ50%
ethyl acetate/hexanes. The product was isolated as a pale yellow solid
(1.0 g, 74%). TLC Rf(50% ethyl acetate/hexanes) = 0.38. Mp = 62 °C.
1H NMR (600 MHz, CDCl3) δ: 6.98 (m, 1 H, ArꢀH), 6.92 (m, 2 H,
ArꢀH), 6.85 (m, 1 H, ArꢀH), 3.85 (s, 3 H, OCH3), 3.09 (s, 4 H,
NCH2), 2.64 (s, 4 H, NCH2), 2.50 (t, 2 H, CH2, J = 4.0 Hz), 2.25 (td,
2 H, CH2, J = 7.0 Hz, J = 2.3 Hz), 1.95 (t, 1 H, CH, J = 2.6 Hz), 1.75 (m,
2 H, CH2). 13C{1H} NMR (125 MHz, CDCl3) δ: 152.3, 141.5, 122.9,
121.1, 118.3, 111.3, 84.3, 68.5, 57.5, 55.4, 53.5, 50.7, 25.9, 16.5.
IR (KBr, cmꢀ1): ν 3295, 2149, 1501. HRMS (CIþ) m/z for
C16H22N2O: calculated, 259.1810; observed, 259.1812 [M þ H]þ.
1-[(4-(2-Methoxyphenyl)piperazin-1-yl)methyl]-1,2-dicarba-
closo-dodecaborane (3). A solution of compound 1 (0.33 g,
1.4 mmol) in toluene (1.5 mL) was added to a suspension of 1-butyl-3-
methylimidazolium chloride (0.050 g, 0.29 mmol) and decaborane
(0.071 g, 0.58 mmol) in toluene (1 mL). The heterogeneous mixture
was heated at reflux (110 °C) for 2 h. The mixture was concentrated to a
viscous yellow liquid using a rotary evaporator. The product was purified
by preparative TLC and an eluent of 100% dichloromethane. The
product was obtained following extraction of the silica with 10%
methanol/dichloromethane and evaporation under reduced pressure
to yield a white solid (0.07 g, 35%). TLC Rf(100% dichloromethane) =
0.79. Mp = 133ꢀ135 °C. 1H NMR (500 MHz, CDCl3) δ: 7.02 (m, 1 H,
ArꢀH), 6.92 (m, 2 H, ArꢀH), 6.86 (m, 1 H, ArꢀH), 4.02 (s, 1 H, CH),
3.85 (s, 3 H, OCH3), 3.12 (s, 6 H, NCH2, CH2), 2.80 (s, 4 H, NCH2).
13C{1H} NMR (125 MHz, CDCl3) δ: 152.2, 140.8, 123.2, 120.9, 118.2,
111.3, 74.9, 62.1, 58.2, 55.4, 54.9, 50.7. 11B{1H} NMR (160 MHz,
CDCl3) δ: ꢀ4.1, ꢀ6.5, ꢀ10.1, ꢀ12.5, ꢀ13.6, ꢀ14.3. IR (KBr, cmꢀ1):
ν 2576, 1500. HRMS (CIþ) m/z for C14H28N2OB10: calculated,
350.3132; observed, 350.3109 [Mþ].
Reactions were performed under an inert atmosphere unless other-
wise stated. All solvents were obtained from Caledon and either dried by
a Pure-Solv solvent purification system (Innovative Technology Inc.) or
dried over calcium hydride (acetonitrile, dichloromethane, and toluene),
sodium/benzophenone (diethyl ether and tetrahydrofuran), or sodium
iodide (acetone) and distilled prior to use. Deuterated solvents for NMR
samples were purchased from Cambridge Isotope Laboratories. Tech-
netium-99m complexes were prepared from pertechnetate [99mTcO4]ꢀ
which was obtained from a 99 Moꢀ99mTc generator (Lantheus Medical
Imaging) in saline (0.9% NaCl).
Reactions were monitored using Alugram Sil G/UV254 thin-layer
chromatography (TLC) plates. Carborane-containing species were visua-
lized with 0.2% PdCl2 in hydrochloric acid (3.0 M), which upon heating
gavedark brown spots. Amineswere visualizedwithninhydrinspray. Silica
gel 60PF254 containing gypsum (EM Science) was used for making
preparative TLC plates and plates for the Chromatotron model 7924T
(Harrison Research). Column chromatography was accomplished with
silica gel 60 (EMD Chemical Inc.) or Ultra Pure silica gel 60 (Silicycle).
Automated normal and reverse-phase (C18) silica gel chromatography
was performed on a Biotage SP1 purification system operated at ambient
temperature using solvent gradients as specified. Solid phase extraction
cartridges (C18) obtained from Waters were used following pretreatment
with water (10 mL), methanol, or ethanol (10 mL) and a second wash
with water (10 mL) and HCl (10 mM, 10 mL).
Instrumentation. Nuclear magnetic resonance spectra (1H, 13C-
{1H}, 11B{1H}) were recorded on either a Bruker 500 or 600 MHz
spectrometer at ambient temperature. The chemical resonances (δ) are
reported in parts per million (ppm), with the 1H NMR shifts referenced
to the residual proton signal of the deuterated solvent and the 13C shifts
referenced to the carbon signal of the solvent. The chemical shifts (δ) for
11B{1H} NMR spectra were reported relative to an external reference of
BF3 Et2O. Infrared spectra were acquired on a Bio-Rad FTS-40, Nicolet
3
510, or 6700 Fourier transform IR spectrometer at ambient temperature.
Samples were run on a KBr plate or as KBr pellets. A Gallenkamp
melting point apparatus was used to determine melting points. Reac-
tions involving microwave heating were performed on a Biotage Initiator
60 or Initiator 8 microwave reactor using crimped-sealed vials. Mass
spectra were obtained from the McMaster Regional Centre for Mass
Spectrometry on a Micromass Quattro Ultima (LC-ESI/APCI triple
quadrupole) mass spectrometer for electrospray ionization (ES) mass
spectrometry, a Micromass GC-EI/CI time of flight mass spectrometer
instrument for chemical ionization (CI) and electron ionization (EI)
spectra, and a Micromass Global Ultima (MALDI/CapLC-ESI quadru-
pole time of flight) mass spectrometer for high resolution mass spectra.
A Capintec dose calibrator was used to determine the activity of samples
containing technetium-99m (>300 kBq).
Tested compounds were purified to >95% as determined by high
performance liquid chromatography (HPLC), which was performed on
a Varian ProStar model 230 or 230I solvent delivery system fitted with a
Varian ProStar model 230 or 335 PDA detector and an in-line radio-
activity detector (IN/US γ-RAM). The wavelength for detection was set
at 250 or 254 nm, and the dwell time in the γ detector was 0.5 s with a
10 μL loop. A Varian Nucleosil C18 100-5 (250 mm ꢁ 4.6 mm) column
was used with a sample volume of either 100 or 500 μL. The following
solvent gradients were employed: method A (solvent A = acetonitrile
(0.1% formic acid), solvent B = 20 mM ammonium acetate, 1 mL/min)
0ꢀ15 min 65ꢀ100% A, 15ꢀ20 min 100% A; method B (solvent A =
aqueous tetraethylammonium phosphate, pH 2.0ꢀ2.5, solvent B =
methanol, 1 mL/min) 0ꢀ3 min 100% A, 3ꢀ6 min 100ꢀ75% A, 6ꢀ9
min 75ꢀ67% A, 9ꢀ20 min 67ꢀ0% A, 20ꢀ22 min 0% A, 22ꢀ25 min
0ꢀ100% A, 25ꢀ30 min 100% A; method C (solvent A = acetonitrile,
solvent B = water, 1 mL/min) 70:30 A/B isocratic for 15 min; method D
1-[(4-(2-Methoxyphenyl)piperazin-1-yl)propyl]-1,2-dicar-
ba-closo-dodecaborane (4). A solution of decaborane (0.082 g,
0.67 mmol) in toluene (5 mL) was added to a suspension of 1-butyl-3-
methylimidazolium chloride (0.041 g, 0.24 mmol) in toluene (5 mL).
A solution of compound 2 (0.23 g, 0.89 mmol) in toluene (5 mL) was
added to the reaction mixture. The heterogeneous mixture was heated at
reflux (110 °C) for 7 days, then concentrated to a viscous yellow liquid
using a rotary evaporator. The product was purified by using automated
silica gel chromatography (12þM) employing a gradient of 0ꢀ2%
methanol/dichloromethane, giving a beige solid following evaporation
of the solvent (0.033 g, 13%). TLC Rf(1% methanol/dichloromethane)
= 0.12. Mp = 138 °C. 1H NMR (500 MHz, CDCl3) δ: 7.00 (m, 1 H,
ArꢀH), 6.93 (m, 2 H, ArꢀH), 6.86 (m, 1 H, ArꢀH), 3.85 (s, 3 H,
OCH3), 3.60 (s, 1 H, CH), 3.08 (s, 4 H, NCH2), 2.61 (s, 4 H, NCH2),
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2.37 (m, 2 H, CH2), 2.28 (m, 2H, CH2), 1.70 (m, 2 H, CH2). 13C{1H}
NMR (125 MHz, CDCl3) δ: 152.4, 141.4, 123.1, 121.2, 118.3, 111.5,
75.4, 61.5, 57.2, 55.5, 53.5, 50.7, 36.1, 26.7. 11B{1H} NMR (160 MHz,
CDCl3) δ: ꢀ2.7, ꢀ6.2, ꢀ9.7, ꢀ11.9, ꢀ12.5, ꢀ13.5. IR (KBr, cmꢀ1): ν
2589, 1501. HRMS (CIþ) m/z for C16H32B10N2O: calculated,
378.3445, observed 378.3452 [Mþ].
Following the final heating, the mixture was cooled to room temperature
and diluted with water (10 mL). The product was then extracted with
dichloromethane (3 ꢁ 10 mL). The organic fractions were combined, dried
over anhydrous sodium sulfate, filtered, and evaporated to dryness. The
product was isolated as an ivory solid (0.13 g, 82%) by either flash or
automated (12þM) silica gel chromatography and a gradient of 12ꢀ100%
ethyl acetate/hexanes or using a reverse phase automated purification system
and a gradient of 20ꢀ100% acetonitrile/water. TLC Rf(50% ethyl acetate/
hexanes) = 0.24. Mp = 110 °C. 1H NMR (600 MHz, CD2Cl2) δ: 7.12 (m,
1 H, ArꢀH), 6.93 (m, 3 H, ArꢀH), 3.84 (s, 3 H, OCH3), 3.79 (AB, 1 H,
CH2, J = ꢀ13.7 Hz), 3.61 (m, 4 H, NCH2), 3.53 (AB, 1 H, CH2, J = ꢀ13.7
Hz), 3.37 (m, 2 H, NCH2), 3.12 (m, 2 H, NCH2), 1.88 (s, 1 H, CH).
13C{1H} NMR (150 MHz, CD2Cl2) δ: 197.8, 153.1, 138.1, 125.7, 121.5,
119.8, 112.1, 66.8, 57.0, 56.9, 55.8, 48.4, 48.4, 44.1, 28.8. 11B{1H} NMR
(160 MHz, CD2Cl2) δ: ꢀ5.8, ꢀ8.0, ꢀ11.4, ꢀ18.5, ꢀ20.3. IR (KBr, cmꢀ1):
ν 2539, 2005, 1907, 1502. HRMS (ESꢀ) m/z for C17H27N2O4B9Re:
calculated, 609.2353; observed, 609.2366 [Mþ]. UVꢀHPLC: method A,
tR = 15.0 min; method B, tR = 22.6 min.
Sodium rac-1-[(4-(2-Methoxyphenyl)piperazin-1-yl)propyl]-
3,3,3-tricarbonyl-3-rhenium-3,1,2-dicarba-closo-dodecaborate
(9). The procedure employed 0.032 g (0.086 mmol) of compound 4.
Following the final heating, the mixture was cooled to room temperature,
diluted with water (10 mL), and extracted with dichloromethane (3 ꢁ
15 mL). The organic fractions were combined, dried over anhydrous sodium
sulfate, filtered, and evaporated to dryness. The product was isolated as an
ivory solid (0.052 g, 95%) by either flash or automated (12þM) silica gel
chromatography and a gradient of 12ꢀ100% ethyl acetate/hexanes or using
a reverse phase automated purification system and a gradient of 20ꢀ100%
acetonitrile/water. TLC Rf(5% methanol/dichloromethane) = 0.52. Mp =
193 °C. 1H NMR (600 MHz, (CD3)2CO) δ: 7.04 (m, 1 H, ArꢀH), 6.99
(m, 2 H, ArꢀH), 6.91 (m, 1 H, ArꢀH), 3.86 (s, 3 H, OCH3), 3.38 (m, 4 H,
NCH2), 2.81 (s, 4 H, NCH2), 1.91 (m, 6 H, CH2), 1.75 (s, 1 H, CH).
13C{1H} NMR (125 MHz, (CD3)2CO) δ: 200.4, 153.5, 140.4, 124.8, 121.9,
119.5, 113.0, 79.2, 58.0, 55.9, 54.2, 53.2, 48.5, 37.7, 25.4. 11B{1H} NMR (160
MHz, (CD3)2CO) δ: ꢀ5.2, ꢀ7.8, ꢀ9.8, ꢀ11.2, ꢀ12.1, ꢀ18.0, ꢀ19.7. IR
(KBr, cmꢀ1): ν 2542, 1995, 1897, 1500. HRMS (ESꢀ) m/z for
C19H31N2O4B9Re: calculated, 637.2697; observed, 637.2679 [Mþ].
UVꢀHPLC: method A, tR = 14.8 min; method B, tR = 23.4 min.
Sodium rac-N-[7-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-
7,8-dicarba-nido-undecaborate-1-carboxamide (5). To a solu-
tion of 1,2-dicarba-closo-dodecaborane-1-carboxylic acid (1.0 g, 5.3 mmol)
in dichloromethane (50 mL) were added thionyl chloride (1.2 mL,
16 mmol) and N,N-dimethylformamide (0.30 mL, 3.9 mmol). The
solution was heated at reflux for 3.5 h. The solvent was removed under
reduced pressure to yield 6 (1.1 g, 5.3 mmol). Compound 6 was
immediately dissolved in dichloromethane (30 mL) and cooled to 0 °C.
Diisopropylethylamine (1.8 mL, 10 mmol) and compound 7 (2.8 g,
11 mmol) were dissolved in dichloromethane (35 mL) and added to
the reaction vessel. The mixture was allowed to warm to room
temperature and stirred overnight. The mixture was washed with HCl
(0.01 M, 3 ꢁ 30 mL) and brine (30 mL). The organic fraction was dried
over sodium sulfate, filtered, and concentrated under reduced pressure.
The reaction mixture was purified by silica column chromatography
using a gradient of 0ꢀ10% methanol/ethyl acetate. The product was
stirred in an ethanolic solution of sodium hydroxide (5 equiv) overnight,
then concentrated under reduced pressure. The residue was redissolved
in ethyl acetate and washed with HCl (0.1 M), brine, and water. The
organic fraction was concentrated and lyophilized to yield 5 as a white
solid. Yield: 0.4 g, 17%. TLC Rf(10% methanol/dichloromethane) =
0.53. Mp g 230 °C. 1H NMR (500 MHz, (CD3)2CO): δ 7.04 (m, 1 H,
ArꢀH), 6.98 (m, 2 H, ArꢀH), 6.91 (m, 1 H, ArꢀH), 6.65 (s, 1 H, NH),
3.86 (s, 3 H, OCH3), 3.45 (s, 6 H, NCH2), 3.27 (m, 4 H, NCH2, CH2),
2.53 (s, 1 H, CH), 1.89 (m, 2 H, CH2), 1.79 (m, 2 H, CH2), 1.64 (m, 4 H,
CH2). 13C{1H} NMR (125 MHz, (CD3)2CO): δ 173.9, 152.7, 139.6,
123.9, 121.0, 118.8, 112.0, 56.3, 54.9, 54.0, 52.9, 47.7, 40.6, 37.1,
27.1, 20.0. 11B{1H} NMR (160 MHz, (CD3)2CO): δ ꢀ9.6, ꢀ10.6,
14.6, ꢀ17.1, ꢀ17.7, ꢀ21.4, ꢀ23.0, ꢀ32.8, ꢀ36.1. IR (KBr, cmꢀ1):ν 3419,
2916, 2523, 1593. HRMS (ESþ): calculated for C18H35B9N3O2,
424.3567; observed, 424.3593 [Mþ].
4-[4-(2-Methoxyphenyl)piperazin-1-yl]butan-1-amine (7).
The synthesis was adapted from Chu and co-workers.35 To a solution
of compound 14 (4.1 g, 15.8 mmol) in dry diethyl ether (150 mL) at
0 °C, 25 mL of lithium aluminum hydride (1.0 M in Et2O) was added
dropwise with stirring. The reaction mixture was stirred at 0 °C for 10
min, then stirred at room temperature for 2 h and finally heated at reflux
for 2 h. The mixture was cooled to 0 °C, and saturated aqueous NaHCO3
(50 mL) was added slowly. The reaction mixture was stirred for 10 min
at 0 °C and 10 min at room temperature. The mixture was filtered
through Celite, and the filtrate was added to a separatory funnel. The
layers were separated, and the aqueous fraction was further extracted
with dichloromethane (3 ꢁ 20 mL). The combined organic fractions
were dried with anhydrous Na2SO4, filtered and the solvent was
removed to yield compound 7 as a yellow oil (3.8 g, 91%). Character-
ization data matched the data reported in the literature.35
Sodium rac-8-[N-[7-[4-(2-Methoxyphenyl)piperazin-1-yl]
butyl]]-2,2,2-tricarbonyl-2-rhenium-2,1,8-dicarba-closo-do-
decaborate-1-carboxamide (10). The procedure employed 0.30 g
(0.67 mmol) of compound 5. Following the final heating, the solvent
was evaporated and the mixture was suspended in acetonitrile (15 mL)
and filtered. The complex was isolated as a white solid following
purification by reverse phase chromatography using a gradient of
20ꢀ100% acetonitrile/water followed by silica column chromatography
or preparative TLC using a gradient of 10% methanol/dichloromethane
(0.046 g, 10%). TLC Rf(5% methanol/dichloromethane) = 0.31. Mp g
230 °C. 1H NMR (600 MHz, CD3CN) δ: 7.03 (m, 1 H, ArꢀH), 6.94
(m, 3 H, ArꢀH), 6.53 (s, 1 H, NH), 3.83 (s, 3H, OCH3), 3.22 (m, 4 H,
NCH2), 3.12 (m, 6 H, NCH2, CH2), 2.89 (m, 2 H, CH2), 1.86 (s, 1 H,
CH), 1.58 (m, 2 H, CH2), 1.47 (m, 2 H, CH2). 13C{1H} NMR (125
MHz, CD3CN) δ: 199.7, 168.5, 153.4, 141.1, 124.5, 122.0, 119.4, 112.9,
57.7, 56.1, 53.8, 49.2, 39.6, 28.6, 27.3, 22.2. 11B{1H} NMR (160 MHz,
CD3CN) δ: ꢀ5.8, ꢀ7.7, ꢀ8.9, ꢀ12.2, ꢀ18.8, ꢀ19.9. IR (KBr,
CH3CN, cmꢀ1): ν 2517, 2002, 1917, 1896. HRMS (ESꢀ) m/z for
C21H35N3O5B9Re: calculated, 695.2972; observed, 695.2999 [Mþ].
UVꢀHPLC: method A, tR = 13.7 min.
General Preparation of Rhenacarboranes 8ꢀ10. The desired
nido-carborane was generated by heating the corresponding closo-carborane
derivative (3 or 4) with sodium fluoride (7 equiv) in aqueous ethanol
(10ꢀ15%) in a microwave reactor (195 °C, 10ꢀ15 min), while nido-
carborane 5 was used directly. Complexation was accomplished by heating
the [Re(CO)3(H2O)3][Br] (1 equiv) with the nido-carboranes in a micro-
wave reactor at 180ꢀ195 °C for 10 min. Two subsequent additions of
[Re(CO)3(H2O)3][Br] (1.0 and 0.5 equiv) were performed where each
addition was accompanied by heating the mixture at 180ꢀ195 °C for 10 min.
Sodium rac-8-[(4-(2-Methoxyphenyl)piperazin-1-yl)methyl]-
2,2,2-tricarbonyl-2-rhenium-2,1,8-dicarba-closo-dodecaborate
(8). The procedure employed 0.093 g (0.26 mmol) of compound 3.
4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyronitrile (14).
The synthesis was adapted from a report by Chu and co-workers.35
To a solution of 1-(2-methoxyphenyl)piperazine (5.1 g, 26.5 mmol) in
acetonitrile (50 mL), sodium carbonate (5.6 g, 52.8 mmol) and
bromobutyronitrile (2.0 mL, 20.1 mmol) were added with stirring.
The reaction mixture was heated to reflux (82 °C) for 24 h. When the
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Journal of Medicinal Chemistry
ARTICLE
Table 2. X-ray Crystal Data for Compound 10
space with the program MAX3D;39 this indicated a c-glide that otherwise
was not detected by APEX2. The structure was then solved by direct
methods and refined by full matrix least-squares refinement on F2 using
the Bruker SHELXTL program library.40 Non-hydrogen atoms were
refined anisotropically, and hydrogen atoms were located from the
difference map. The hydrogen atoms that were well-behaved were
allowed to refine; others were fixed at calculated positions and were
refined as riding on the atom to which they were bonded. Protonation
of one of the nitrogen atoms in the piperazine ring meant that there was
no residual counterion in the complex. In the final stages of refinement,
R = 3.89% and wR2 = 6.83%; CCDC No. 795365.
parameter
empirical formula
formula weight
crystal system
space group
unit cell parameters
a (Å)
C21H35B9N3O5Re
693.01
monoclinic
P2(1)/c
9.6837(5)
12.8205(7)
23.6507(13)
90
b (Å)
c (Å)
R (deg)
’ ASSOCIATED CONTENT
β (deg)
95.7870(10)
90
S
Supporting Information. Screening and characteriza-
b
γ (deg)
tion data for the ligands and metal complexes. This material is
temp of data collection (K)
Z
173(2)
4
available free of charge via the Internet at http://pubs.acs.org.
R indices (all data)
R1
’ AUTHOR INFORMATION
0.0704
0.0752
1.038
wR2
Corresponding Author
*Phone: 905-525-9140, extension 20182. Fax: 905-522-2509.
E-mail: valliant@mcmaster.ca.
goodness-of-fit
mixture was cooled, anhydrous Na2SO4 was added. The mixture was
filtered and the solvent removed under reduced pressure to yield a
yellow oil. The product was purified using silica column chromatography
using a gradient of 75ꢀ100% ethyl acetate/hexanes (4.6 g, 88%).
Characterization data matched the data reported in the literature.35
General Preparation of 99mTc Carborane Complexes. The
nido-carborane were generated by heating the closo-carborane derivative
3 or 4 with sodium fluoride (7 equiv) in aqueous ethanol (10ꢀ15%) in a
microwave reactor (195 °C for 10 min), while nido-carborane 5 was used
directly. To a solution of the ligand (3, 6 mM; 4, 5 mM; 5, 4 mM),
’ ACKNOWLEDGMENT
The authors thank the McMaster Regional Centre for Mass
Spectrometry, McMaster Analytical X-ray Diffraction Facility,
and Dr. Bryan Roth at the University of North Carolina at Chapel
Hill and the National Institute of Mental Health’s Psychoactive
Drug Screening Program, Contract No. NO1MH32004, for in
vitro analysis. This work was supported by the National Sciences
and Engineering Research Council (NSERC) of Canada. We
thank the Province of Ontario for an OGS-ST scholarship for
A.S.L.
[
99mTc(CO)3(H2O)3]þ (370ꢀ1850 MBq, 1 mL) was added to the
reaction vessel. The mixture was heated in the microwave reactor at
195 °C for 10 min. The crude reaction mixture was passed through a C18
solid phase extraction cartridge where residual [99mTc(CO)3(H2O)3]þ
was eluted with HCl (10 mM) and the product was eluted with
acetonitrile. The excess ligand was removed by HPLC (method A).
Decay corrected radiochemical yield: 36% (11), 19% (12), 14% (13).
Radio-HPLC: method A, tR = 15.7 min (11), 15.2 min (12), 14.0 min
(13); method B, tR = 22.9 min (11), 23.1 min (12).
’ ABBREVIATIONS USED
WAY, 1-(2-methoxyphenyl)piperazine; Cp, cyclopentadiene;
MIP, molecular imaging probe;CNS, central nervous system; PET,
positron emission tomography; SPECT, single photon emission
computed tomography; SAR, structureꢀactivity relationship;
LCꢀMS, liquid chromatographyꢀmass spectrometry; DAT,
dopamine active transporter; SERT, serotonin transporter; NET,
norepinephrine transporter; TLC, thin layer chromatography;
ES, eletrospray; CI, chemical ionization; EI, electron impact;
HPLC, high-performance liquid chromatography
Stability Studies. The 99mTc complexes 11ꢀ13 were dissolved in
ethanol (0.5 mL) and transferred to a vial containing saline (0.9% NaCl,
4.5 mL). Analytical HPLC of the samples was performed every 30 min
using a Varian Nucleosil C18 column (methods CꢀG). No decom-
position was observed by radio-HPLC over 6 h under several different
analytical HPLC conditions (mobile phase and pH).
In Vitro Assay. Ki determination was conducted by the National
Institute of Mental Health’s Psychoactive Drug Screening Program,
Contract No. NO1MH32004. For experimental details refer to the
PDSP Web site http://pdsp.med.unc.edu/. Compounds 8ꢀ10 were
assayed against serotonin, R- and β-adrenergic, DAT, NET, SERT, and
σ receptors.
X-ray Analysis. A colorless rod-shaped crystal of 10 was mounted
on a MiTeGen polymer mount and placed in a cold stream of liquid
nitrogen (77 K). Data (Table 2) were collected at 100 K on a Bruker
APEX2 diffractometer equipped with CCD area detector using a
graphite monochromator Mo KR radiation and φ and ω scans. The
APEX2 suite was used for data collection, and SAINT was used for data
reduction; data were scaled using SADABS with both face-indexed
absorption correction and a correction using redundant data.36ꢀ38 The
correct space group (P21/c) was chosen by examination of reciprocal
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