Target-specific ligands and gadolinium-based complexes for imaging of dopamine receptors: Synthesis, binding affinity, and relaxivity

Article abstract of DOI:10.1021/jo400646k

Magnetic resonance imaging (MRI) and positron emission tomography (PET) are two extremely important imaging modalities with unlimited tissue penetration. Molecular imaging is a field by which specific targets or biological processes are imaged. MRI, which is used for functional imaging and for the diagnosis of a broad range of pathologic conditions, suffers from limited specificity and intrinsically low sensitivity. One possibility to alleviate partially these limitations is to use contrast agents (CAs) and more importantly target-specific CAs. We have developed a modular synthesis of novel ligands and gadolinium(III)-based target-specific MRI CAs with high relaxivity and high binding affinity toward the dopamine receptors. The prepared ligands and MRI CAs are based on spiperone as targeting moiety. The prepared target-specific CAs can potentially be used for in vitro and possibly in vivo MR imaging of dopaminergic receptors. Importantly the ligands prepared using the modular approach presented in this paper may also be useful for other imaging modalities such as PET (or SPECT) by just replacing, at the last stage of the synthesis, the gadolinium cation by other metal cations having relatively long half-lives, such as ⁶⁴Cu, ⁸⁹Zr, ¹¹In, and more.

Full text of DOI:10.1021/jo400646k

Article
pubs.acs.org/joc
Target-Specific Ligands and Gadolinium-Based Complexes for
Imaging of Dopamine Receptors: Synthesis, Binding Affinity,
and Relaxivity
Isaac Zigelboim, Avi Weissberg, and Yoram Cohen*
School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv,
Tel-Aviv 69978, Israel
S
* Supporting Information
ABSTRACT: Magnetic resonance imaging (MRI) and positron
emission tomography (PET) are two extremely important
imaging modalities with unlimited tissue penetration. Molec-
ular imaging is a field by which specific targets or biological
processes are imaged. MRI, which is used for functional imag-
ing and for the diagnosis of a broad range of pathologic con-
ditions, suffers from limited specificity and intrinsically low
sensitivity. One possibility to alleviate partially these limita-
tions is to use contrast agents (CAs) and more importantly
target-specific CAs. We have developed a modular synthesis of
novel ligands and gadolinium(III)-based target-specific MRI
CAs with high relaxivity and high binding affinity toward the dopamine receptors. The prepared ligands and MRI CAs are based
on spiperone as targeting moiety. The prepared target-specific CAs can potentially be used for in vitro and possibly in vivo MR
imaging of dopaminergic receptors. Importantly the ligands prepared using the modular approach presented in this paper may
also be useful for other imaging modalities such as PET (or SPECT) by just replacing, at the last stage of the synthesis, the
gadolinium cation by other metal cations having relatively long half-lives, such as 64Cu, 89Zr, 11In, and more.
of “smart” gadolinium CAs for imaging pH, temperature,
and different metals,7a,8,9 MR imaging of specific receptors
remained a formidable challenge.10 Nevertheless, during the
past decade a few elegant demonstrations of the feasibility of
this approach, using MRI methodology, have been reported.11
These included, inter alia, an elegant example for indirect MR
imaging of gene expressions11a,b and imaging of the engineered
version of the transferring receptor on the surface of 9L glioma
cells.11c These examples served as the first proof of concept for
the potential uses of MRI in molecular imaging.11a−c More
recently new transgene reporters for in vivo MRI have been
reported, and some gadolinium-based target-specific CAs were
shown to operate even in vivo, most of them in cancer cells and
tumors where specific receptors are overexpressed.12 In
contrast, PET, which has much higher sensitivity than MRI,
has been used extensively in molecular imaging in general and
for imaging of specific receptors in particular.1b The main
drawback of PET is that the methods rely on the use of
radioactive tracers for achieving these goals.
INTRODUCTION
■
Magnetic resonance imaging (MRI) and positron emission
tomography (PET) are two of the most important imaging
modalities in biomedicine and in the clinics.1 Molecular
imaging, the field aimed at visualizing specific molecular targets
or biological processes, is gaining increasing attention in recent
years.2 MRI is a very valuable imaging technique for studying
tissue functions and for the diagnosis of a broad range of
pathologies.1a MRI has many advantages compared to other
imaging techniques:1a,3 non-invasiveness, high tissue penetra-
tion, and relatively high temporal and spatial resolution.1a
In addition, MRI uses a variety of physical parameters as
contrast mechanisms and can be combined with MR spectros-
copy (MRS) of metabolites. However, the major drawbacks
of MRI are the limited specificity and the intrinsically low
sensitivity of the technique.1a
One of the main possibilities to increase MRI specificity
and, to a limited extent, the method’s low sensitivity is to use
contrast agents (CAs) with specific tissue distribution. Indeed
such CAs, which first were mostly intravascular, have been pre-
pared in the past three decades.4 A more challenging approach
to increase MRI specificity, however, is to design target-specific
MRI CAs5 with high affinity to a specific molecular target,
thus expanding the scope of MRI into the field of molecular
imaging.6,7 Such approach may, in principle, open the way for
indirect MR imaging of specific receptors, enzymes, and other
biological processes. Despite the recent progress in the design
In the present work we decided to develop a modular syn-
thesis of ligands that have the potential to be used as target-
specific CAs for imaging of the dopamine receptors (DRs) by
MRI or PET. We selected the D3/D2 DRs as the target since
the dopaminergic system is believed to be relevant to many
Received: April 18, 2013
Published: June 18, 2013
© 2013 American Chemical Society
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a
Scheme 1. Synthesis of Compounds 9a and 9b and Their Respective Gadolinium Complexes 10a and 10b
a
(i) 10 equiv Diisopropylamine (DIPA) in DMF at 78−80 °C for 18 h; (ii) 1.5 equiv KOH in dry DMF at 120 °C for 18−20 h; (iii) 2 equiv TsCl with 4-
Dimethylaminopyridine (DMAP; cat. amount) in dry Dichloromethane (DCM) with 8 equiv Triethylamine (TEA) at 0−10 °C for 3 h; (iv) 5 equiv
Potassium carbonate (K2CO3) in acetonitrile (ACN) at 80 °C for 6 h; (v) Trifluoroacetic acid (TFA, excess) at rt for 4 h; (vi) GdCl3 in H2O with MeOH.
neurological diseases and therefore attracts much therapeutic
interest.13 These receptors were identified, inter alia, as the
primary sites of action for most antiparkinsonic and
antipsychotic drugs. The DRs have relative high concentration,
which in some areas of the normal human brain is about
10 nM.13d In addition, the D3/D2 DRs, which are known to be
the second most abundant DR type in the mammalian brain,
are also known to have an uneven distribution in the brain.13
Keeping these facts in mind and observing the wide range of
different ligand types that can bind DRs,14 we decided to design
novel ligands and gadolinium-based target-specific MRI CAs for the
dopamine receptors. Previously we prepared CAs based on the
“spiro” part of the well-known dopaminergic ligand spiperone.15,16
The R1 and R2 relaxivities of the obtained compounds were found
to be higher than those of Gd3+-DOTA. However, their binding
affinities to the dopamine receptor were too low.16 It is clear
that any CAs aimed at imaging receptors should have a high
binding affinity to the target receptor. To improve the binding
affinity of our CAs to the dopamine receptors and to get more
information on the structure−affinity relationship (SAR) in the
binding of such systems, we synthesized a series of ligands from
which we prepared gadolinium-based target-specific CAs that
contain spiperone or its etheric analogue, AMI-193, as the bind-
ing moieties and studied their relaxivity and binding affinity.
RESULTS AND DISCUSSION
■
Ligands 9a and 9b: Synthesis and Characterization. In
our design the dopamine receptor binding moieties of the
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Figure 1. (A) HRMS (TOF MS ES negative), (B) 19F NMR (376.3 MHz; CD3OD, 25 °C), and (C) 1H NMR (400 MHz; CD3OD, 25 °C) spectra of 9a.
ligands and the gadolinium-based CAs prepared thereof were
derived from 8-[4-(4-fluorophenyl)-4-oxo-butyl]-1-phenyl-
1,3,8-triazaspiro[4,5]decan-4-one, known also as spiperone
(3a in Scheme 1), a strong ligand of the dopamine D3/D2
dopamine receptor17a or its etheric analogue 8-[3-(4-
fluorophenoxy)propyl]-1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one,
known also as AMI-193 (3b in Scheme 1).17b,c The
paramagnetic moiety of the designed target-specific CAs is a
Gd3+-complex of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-
(acetate), known as DO3A, while the spacers were based on
alkane and tri- and tetra(ethylene glycol) (triEG and tetraEG)
moieties.
used by us previously.16 Compounds 3a and 3b were then
N-alkylated at position 3 by 11-bromo-1-undecanol (4) under
basic conditions to afford the alcohols 5a and 5b in 81% and
89% yield, respectively. These alcohols were then tosylated in
presence of N,N-dimethylaminopyridine to give the respective
tosylates 6a and 6b in 80% and 74% yield, respectively. Sub-
sequently compounds 6a and 6b were reacted with the com-
mercially available compound 7 to give the triesters 8a and 8b
in 70% and 76% yield, respectively. The triesters 8a and 8b
were then nearly quantitatively deprotected by TFA to afford
the ligands 9a and 9b as a penta-salt. These ligands were char-
1
acterized by H, 13C, and 19F NMR and high resolution mass
The ligands (9a and 9b) and the gadolinium-based CAs with
undecylenic spacers (10a and 10b in Scheme 1) were syn-
thesized from spiperone and AMI-193 (3a and 3b in Scheme 1,
respectively) in five steps with an overall yield of about 50% as
outlined in Scheme 1. The starting compounds 3a and 3b were
synthesized in high yields (of about 90%) by N-alkylation of the
“spiro” compound 1-phenyl-1,3,8-triazaspiro[4,5]decan-4-one
(1) at position 8 by the respective halides (2a and 2b) under
mild basic conditions, using a procedure similar to the one
spectrometry (see Figures 1 and 2 and Figures S33−S37 in the
Supporting Information).
Ligands 15a−d: Synthesis and Characterization.
Because we observed that the water solubility of ligands 9a
and 9b is not very high, we decided to prepare also a series
of ligands with water-soluble spacers (see Scheme 2). We chose
to prepare ligands 15a−d that have water-soluble tri- and
tetra(ethylene glycol) (EG) spacers. It should be noted that the
ethylene glycol spacers may also make the compounds more
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Figure 2. (A) HRMS (TOF MS ES negative), (B) 19F NMR (376.3 MHz; CD3OD, 25 °C), and (C) 1H NMR (400 MHz; CD3OD, 25 °C) spectra of 9b.
biocompatible.19 The detailed synthesis of these ligands and the
gadolinium complexes prepared thereof outlined in Scheme 2
shows that ligands 15a−d were synthesized in five steps with an
overall yields of about 45%.
both PET and SPECT are much more sensitive than MRI and
hence are more suitable for imaging of receptors, both tech-
niques use radioactive tracers, so we decided to concentrate on
the preparation of gadolinium-based CAs suitable for MRI.
Gadolinium-Based Complexes: Synthesis and Charac-
terization. Ligands 9a,b were quantitatively converted to their
respective gadolinium complexes 10a,b by a known literature
procedure (Scheme 1).19 Clear and conclusive evidence for the
formation of the paramagnetic complexes 10a and 10b was ob-
tained from high-resolution mass spectrometry (HRMS) (see
Figure 3 and Supporting Information).
The same procedure was used to convert compounds 15a−d
to their Gd3+ paramagnetic complexes 16a−d (Scheme 2).
Clear evidence for the formation of the paramagnetic com-
plexes was obtained from HRMS (TOF MS ESI) spectra (see
Figure 4 and Supporting Information).
To obtain compounds 15a−d, 3a and 3b were first reacted with
1.3 equiv of the respective oligo-EG monotosylate 11a20 or 11b20
in DMF with 1.3 equiv of KOH at 110 °C to afford alcohols
12a−d, respectively in yields of over 70% (see Scheme 2). Com-
pounds 12a−d were then tosylated to give the respective tosylates
13a−d, which were then reacted with the macrocyclic intermediate
7 affording the triesters 14a−d, respectively. Compounds 14a−d
were deprotected by TFA to afford the penta-salts 15a−d,
respectively, and their full spectroscopic characterization is
shown in Figures S93−S105 in Supporting Information.
The prepared ligands can be used to prepare metal-cation-
based CAs like those used in PET, SPECT, and MRI. Although
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a
Scheme 2. Synthesis of Compounds 15a−d and Their Respective Gadolinium Complexes 16a−d
a
(i) 1.3 equiv oligoEG-monotosylate in DMF with 1.3 equiv KOH at 110 °C for 15 h; (ii) 2 equiv TsCl with DMAP (cat. amount) in dry DCM with 6 equiv
TEA at 10 °C to rt for 3 h; (iii) 5 equiv K2CO3 in ACN at 80 °C for 4 h; (iv) TFA (excess) at rt for 4 h; (v) GdCl3 in H2O.
Figure 3. HRMS (TOF MS ES positive) of the isotope distribution of the experimental molecular peaks of (A) 10a and (B) 10b.
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Figure 4. (A) HRMS (TOF MS ES negative) of the isotope distribution of molecular peak of 16a and (B) HRMS (TOF MS ES positive) of the
isotope distribution of the calculated and experimental molecular peak of 16b.
Binding Affinity to the Dopamine Receptor and
Relaxivity of the Target-Specific CAs 10a,b and 16a−d.
As a first stage we tested the binding affinity of our gadolinium-
based CAs to the dopamine receptors and then evaluated their
relaxivity (longitudinal relaxivity R1 and transverse relaxivity R2).
To assess the affinity of the obtained target-specific CAs to the
D3/D2 DRs, we have performed an essay of [3H]-spiperone
binding [5 × 10−10 M] to isolated mouse striatal membranes as we
described previously.16,21 In our essay we measured the concentra-
tion that induces 50% inhibition as compared to H-spiperone.
The binding affinity values of 10a and 10b were found to be
much higher than the previously reported ones for the “spiro”-
based CAs, which were previously found to be about
(10−5−10−6 M; see Table 1).16 The binding results for the
AMI-193-based CAs, however, were less satisfying. Indeed, 16d
was found to have a binding affinity close to that previously
reported for the “spiro”-based compound with the relatively
long (C8) spacer.16 The spiperone-based CAs 10a and 16b,
however, were found to have very high binding affinities toward
the dopamine receptors (close to that of spiperone itself). Thus,
3
the 50% inhibition of H-spiperone binding (5 × 10−10 M) was
obtained at concentrations of 1 × 10−8 M and 5 × 10−9 M for
16b and 10a, respectively (see Table 1).
The results obtained in the present study together with those
reported previously16 shed light on the structural requirements
to obtain high affinity ligands and complexes for dopamine
21
3
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Table 1. Comparative Binding Affinity (50% Inhibition Concentration) and Relaxivity (R1 and R2 Values) of Spiperone-Based
(10a and 16b), AMI-193-Based (16d), and Previously Prepared “Spiro”-Based (C3-Spacered and C8-Spacered) CAs16
10a
16b
16d
“spiro”-based C3-spacered CA16
“spiro”-based C8-spacered CA16
D2 binding affinity [M]
5 × 10−9
5.5
1 × 10−8
7.8
10−6
10−5
10−6
R1 [mM−1 s−1
R2 [mM−1 s−1
]
]
a
a
5.9
8.3
23.6
28.8
18.1
22.6
a
Because of the low binding affinity of 16d to the DRs its R1 and R2 were not be measured.
receptors in the spiperone family. Our results show that the
“spiro” part of the spiperone is not sufficient for effective
binding to the dopamine receptors. Even changing the carbonyl
group of the spiperone to an etheric moiety resulted in a much
lower affinity toward the dopamine receptors. Indeed, despite the
many different ligands that bind strongly to the DRs,
our results clearly show that the entire spiperone is needed for
effective binding to these receptors and that binding of even a large
chemical moiety through the N3 position (see Scheme 1) of the
spiperone does not reduce considerably the binding affinity to the
dopamine receptors. This indeed indicates that the N3 position is
the best position to modify the spiperone moiety without
compromising the binding affinity to receptors. It is interesting to
note also that compound 10a with a hydrophobic C11 spacer has
higher binding affinity than its analogue 16b with a hydrophilic
tetraEG spacer (see Table 1). For the target-specific gadolinium
complexes that were found to have high binding affinities to the
dopamine receptors (10a and 16b), we evaluated also their
relaxivity. The R1 and R2 relaxivity of 10a were found to be 5.5 and
23.6 mM−1 s−1, respectively, while those of 16b were found to be
7.8 and 28.8 mM−1 s−1, respectively. These values are somewhat
higher than those reported previously for the “spiro”-based CAs16
and are also significantly higher than those of Gd-DOTA.22
It is interesting to note that the R1 of 10a is lower than the
R1 of 16b. One possible explanation for this observation may
well be that the long chain connecting the spiperone moiety in
10a is not fully extended in water or may even be folded so that
water has less access to the Gd3+ in 10a as compared to 16b. It
is even more important to note that the R2 relaxivities of both
10a and 16b are higher than 20 mM−1 s−1. These values imply
that these target-specific MR gadolinium complexes may also
be used as “negative” or T2/T2* contrast agents.
To image specific receptors that are not overexpressed by
MR methods is rather challenging and requires not only high
biding affinity to the targets but also significant amplification.
Therefore it is very important that our modular synthesis
provides ligands (9a and 15b) that can, relatively easily, be
transformed into positron PET and SPECT active CAs by just
introducing, in the last step of the synthesis, other metal cations
having relatively long half-lives, such as 64Cu, 111In, 99Tc, and
more.23 This will transform our new ligands into target-specific
PET agents that are extremely important by themselves.1
Because of the much higher sensitivity of PET and SPECT as
compared to MRI, it is clear that our ligands should enable one
to map the dopamine receptors as in the case of 11C-methyl-
spiperone.17 The much longer half-lives of the metal cation
PET agents as compared to 11C PET agents (20 min compared
to 12.7 h and 3 days in the case of 64Cu and 89Zr, respectively,
for example) imply that our new ligands, 9a and 15b, may also
open new avenues of applications in the field of PET imaging.
gadolinium-based MRI CAs all by an efficient modular syn-
thesis. The target-specific gadolinium-based complexes were
found to have high R1 and R2 relaxivity. Most importantly the
spiperone-based CAs were found to have excellent binding
affinity to dopamine receptors, while their “spiro”- and even
AMI-193-based analogues did not. Therefore, the spiperone-
based CAs (10a, 16a, and 16b) have the potential to be used as
target-specific MRI contrast agents, which may enable in vitro
and possibly in vivo MR mapping of dopamine receptors in
experimental models. In vitro MRI experiments along these
lines are now underway. The binding affinity of the different
complexes prepared shed light on crucial structural require-
ments to preserve high binding affinity and enabled us to
identify the structural modifications allowed to maintain strong
binding to the dopamine receptors. Importantly the modular
approach presented in the present paper should, in principle,
allow the preparation of target-specific CAs to be used by other
imaging modalities such as PET and SPECT, by just replacing
the gadolinium cation, in the last step of the synthesis, by other
cations such as 64Cu, 89Zr, 111In, 99Tc, and more. This will enable
mapping of the dopamine receptors with PET and SPECT
tracers having long half-lives.
EXPERIMENTAL SECTION
■
General. [3H]-Spiperone (91 Ci/mmol) was purchased from
Amersham Bio Science UK. 1H, 19F, and 13C NMR spectra and T1 and
T2 relaxation experiments were all acquired on a 400 MHz (9.4T) NMR
spectrometer operating at 400.13, 376.3, and 100.6 MHz for 1H, 19F, and
13C, respectively. MS were obtained by FAB-MS, and the HRMS were
collected on a time-of-flight analyzer (TOF) (ionization mode ESI).
Preparation of 3a and 3b. To a solution of 1 (1.04 g, 4.5 mmol,
1 equiv) in dry dimethylformamide (DMF; 25 mL) and dry diisopropyl-
amine (DIPA; 4.5 g, 6.4 mL, 45 mmol, 10 equiv) was added 4-chloro-4′-
fluorobutyro-phenone (2a) (4.5 g, 3.7 mL, 22.5 mmol, 5 equiv) or 3-(4-
fluorophenoxy)propyl chloride (2b) (4.2 g, 3.5 mL, 22.4 mmol, 5 equiv)
under argon. After stirring for 17 h at 80 °C the reaction mixture was
cooled to room temperature (rt) and then was poured into water
(50 mL). Thereafter, the aqueous phase was extracted with chloroform
(4 × 50 mL). The combined organic phase was dried with magnesium
sulfate, filtered, and evaporated under reduced pressure (first, the
chloroform was evaporated with an evaporator, and then DMF in a
high vacuum system with liquid nitrogen). The obtained grayish
powdery solid was purified by trituration from methanol or by column
chromatography over silica starting with chloroform as eluting solvent
and changing it to 5:95 methanol/chloroform to afford 3a as white
1
powdery solid; yield 1.66 g (4.2 mmol, 93%); mp 201−202 °C; H
NMR (400 MHz, CDCl3, 25 °C) δppm 8.00−8.04 (m, 2H), 7.24−7.28
(m, 2H), 7.13 (t, J = 8.6 Hz, 2H), 6.85−6.91 (m, 3H), 4.73 (s, 2H),
3.02 (t, J = 7.1 Hz, 2H), 2.70−2.90 (m, 4H), 2.40−2.70 (m, 4H), 1.98
(t, J = 7 Hz, 2H), 1.68−1.76 (m, 2H); 13C NMR (100.6 MHz, CDCl3,
25 °C) δppm 198.6, 177.9, 165.6 (d, J = 254 Hz), 143.1, 133.6, 130.7
(d, J = 9 Hz), 129.2, 119.1, 115.7, 115.6 (d, J = 21 Hz), 59.4, 59.2,
57.5, 49.6, 36.4, 29.1, 21.8; 19F NMR (376.3 MHz, CDCl3, 25 °C)
δppm −106.1; FAB+-MS m/z 396.2 [M + H]+.
CONCLUSION
■
Compound 3b was obtained as a white powdery solid; yield 1.50 g,
1
We designed and prepared a series of spiperone and AMI-
193-targeted ligands from which we prepared a series of
(3.9 mmol, 87%); mp 171−172 °C; H NMR (400 MHz, CDCl3,
25 °C) δppm 7.25−7.29 (m, 2H), 6.83−6.99 (m, 7H), 6.59 (bs, NH,
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1H), 4.74 (s, 2H), 4.021 (t, J = 6.4 Hz, 2H), 2.79−2.85 (m, 4H),
2.58−2.70 (m, 4H), 1.96−2.00 (m, 2H), 1.70−1.78 (m, 2H); 13C
NMR (100.6 MHz, CDCl3, 25 °C) δppm 177.8, 157.1 (d, J = 238 Hz),
155.2, 143.2, 129.2, 119.1, 115.8 (d, J = 23 Hz), 115.7, 115.4 (d, J = 8
Hz), 67.0, 59.4, 59.1, 54.9, 49.7, 29.2, 27.1; 19F NMR (376.3 MHz,
CDCl3, 25 °C) δppm −124.8; FAB+-MS m/z 384.1 [M + H]+.
Preparation of 5a and 5b. To a solution of 3a (1.00 g,
2.53 mmol, 1 equiv) or 3b (0.97 g, 2.53 mmol, 1 equiv) in dry DMF
(30 mL) and potassium hydroxide (KOH; 0.21 g, 3.80 mmol, 1.5 equiv)
was added 11-bromo-1-undecanol (4) (0.76 g, 3.04 mmol, 1.2 equiv)
under argon. After stirring for 18−20 h at 120 °C the reaction mixture
was cooled and poured into water (70 mL). Thereafter, the aqueous
phase was extracted with dichloromethane (DCM; 3 × 70 mL). The
combined organic phase was dried with magnesium sulfate, filtered,
and evaporated under reduced pressure (first, the dichloromethane
was evaporated in an evaporator, and then DMF in a high vacuum
system with liquid nitrogen). The residue was purified by column
chromatography over silica. The eluting chloroform solvent was
changed to 3:97 methanol/chloroform (Rf of the product was 0.2) to
afford 5a as clear very viscous liquid; yield 1.16 g (2.05 mmol; 81%);
1H NMR (400 MHz, CDCl3, 25 °C) δppm 8.00−8.04 (m, 2H), 7.23−
7.27 (m, 2H), 7.10−7.15 (m, 2H), 6.82−6.90 (m, 3H), 4.66 (s, 2H),
3.63 (t, J = 6.6 Hz, 2H), 3.41 (t, J = 7.3 Hz, 2H), 3.02 (t, J = 7.2 Hz,
2H), 2.8−2.9 (bm, 4H), 2.6 (bm, 2H), 2.5 (bm, 2H), 1.93−1.99 (m,
2H), 1.52−1.66 (m, 6H), 1.25−1.35 (bm, 14H); 13C NMR (100.6
MHz, CDCl3, 25 °C) δppm 198.6, 174.3, 165.6 (d, J = 254 Hz), 143.0,
133.6, 130.7 (d, J = 9 Hz), 129.2, 118.8, 115.6 (d, J = 22 Hz), 115.2,
63.4, 63.0, 60.7, 57.6, 49.6, 40.8, 36.4, 32.8, 29.47, 29.40, 29.36, 29.35,
29.35, 29.10, 27.3, 26.7, 25.7, 21.9; 19F NMR (376.3 MHz, CDCl3,
25 °C) δppm −106.2; FAB+-MS m/z 537.3 [M + H]+.
2H), 2.86 (m, 4H), 2.58−2.70 (m, 4H), 2.44 (s, 3H), 1.95−2.05 (m,
2H), 1.58−1.74 (m, 6H), 1.22−1.32 (m, 14H); 13C NMR (100.6
MHz, CDCl3, 25 °C) δppm 174.3, 157.1 (d, J = 238 Hz), 155.1, 144.6,
143.0, 133.3, 129.7, 129.2, 127.8, 118.8, 115.7 (d, J = 23 Hz), 115.4 (d,
J = 8 Hz), 115.2, 70.6, 67.0, 63.4, 60.6, 54.9, 49.7, 40.8, 29.39, 29.37,
29.35, 29.31, 29.27, 29.1, 28.86, 28.79, 27.3, 26.7, 25.3, 21.6; 19F NMR
(376.3 MHz, CDCl3, 25 °C) δppm −124.8; FAB+-MS m/z 708.2 [M + H]+.
Preparation of 8a and 8b. 1,4,7,10-Tetraazacyclododecane-1,4,7-
tris(tert-butylacetate) (7) (0.36 g, 0.70 mmol, 1 equiv) was added
to a stirred solution of 6a (0.50 g, 0.70 mmol, 1 equiv) or 6b (0.50 g,
0.70 mmol, 1 equiv) and K2CO3 (0.48 g, 3.50 mmol, 5 equiv) in
acetonitrile (ACN; 10 mL). The solution was heated to 80 °C for 6 h.
The precipitate was filtered, and the filtrate was evaporated under
reduced pressure. The residue was purified by column chromatog-
raphy over silica. The eluting solvent was changed from chloroform to
10% methanol in chloroform (Rf of the product was 0.5) to afford 8a
1
as a very viscous liquid; yield 0.52 g (0.49 mmol, 70%); H NMR
(400 MHz, CDCl3, 25 °C) δppm 7.95−8.01 (m, 2H), 7.79 (d, J = 8.0 Hz,
2H; tosylate anion), 7.19 (t, J = 7.9 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H),
7.06 (d, J = 8.2 Hz, 2H; tosylate anion), 6.85 (d, J = 8.3 Hz, 2H), 6.80
(t, J = 7.3 Hz, 1H), 4.63 (s, 2H), 3.33 (t, J = 7.3 Hz, 2H), 1.90−3.33
(bm, 41H; includes s of tosylate anion), 1.10−1.80 (bm, 45H); 13C
NMR (100.6 MHz, CDCl3, 25 °C) δppm 198.3, 174.0, 173.4, 172.5,
165.9 (d, J = 254 Hz), 144.2, 142.7 (tosylate anion), 138.4 (tosylate
anion), 133.4, 130.6 (d, J = 9 Hz), 129.2, 128.2 (tosylate anion), 126.2
(tosylate anion), 118.8, 115.5 (d, J = 22 Hz), 115.1, 82.4, 82.1, 63.3,
60.2, 57.3, 56.2, 55.6, 54.3, 50.2, 49.4, 40.7, 36.2, 29.5, 29.5, 29.4, 29.4,
29.1, 28.9, 27.82, 27.83, 27.83, 27.83, 27.84, 27.7, 27.7, 27.7, 27.2,
26.7, 21.2 (tosylate anion); 19F NMR (376.3 MHz, CDCl3, 25 °C)
δppm −106.1; FAB+-MS m/z 1084.7 [M + Na]+.
Compound 8b was obtained as a very viscous liquid; yield 0.56 g
(0.53 mmol, 76%); 1H NMR (400 MHz, CDCl3, 25 °C) δppm 7.82 (d,
J = 8.0 Hz, 2H; tosylate anion), 7.25 (t, J = 8.0 Hz, 2H), 7.10 (d, J =
8.1 Hz, 2H; tosylate anion), 6.89−7.00 (m, 4H), 6.80−6.87 (m, 3H),
4.68 (s, 2H), 4.01 (t, J = 6.1 Hz, 2H), 3.42 (t, J = 7.4 Hz, 2H), 3.0−3.2
(bm, 8H), 2.77 (bs, 8H), 2.2−2.4 (bm, 9H; includes s of tosylate
anion), 2.08 (bs, 2H), 1.66−1.73 (m, 2H), 1.59−1.65 (m, 2H), 1.20−
1.55 (m, 53H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 174.0,
173.4, 172.6, 157.1 (d, J = 238 Hz), 154.9, 143.9, 142.6 (tosylate
anion), 138.6 (tosylate anion), 129.3, 128.3 (tosylate anion), 126.1
(tosylate anion), 118.9, 115.7 (d, J = 23 Hz), 115.4 (d, J = 8 Hz),
115.0, 82.5, 82.2, 77.2, 66.6, 63.4, 60.1, 56.3, 55.6, 54.9, 54.3, 50.2,
49.5, 40.8, 29.6, 29.44, 29.42, 29.1, 28.8, 28.1, 28.0, 27.9, 27.9, 27.8,
27.7, 27.5, 27.3, 26.7, 26.6, 21.2 (tosylate anione); 19F NMR (376.3
MHz, CDCl3, 25 °C) δppm −124.6; FAB+-MS m/z 1072.5 [M + Na]+.
Preparation of 9a and 9b. Compound 8a or 8b was dissolved in
trifluoroacetic acid (TFA). After stirring at ambient temperature for
4 h the solution was evaporated in vacuum. Methanol was added and
evaporated, and then chloroform was added and evaporated (until the
complete disappearance of TFA). Compound 9a was obtained nearly
quantitatively as a light brown viscous substance; 1H NMR (400 MHz,
CD3OD, 25 °C) δppm 7.91−7.97 (m, 2H), 7.68 (d, J = 8.1 Hz, 2H;
tosylate anion), 7.32 (t, J = 7.9 Hz, 2H), 7.10−7.28 (m, 4H; includes d
of tosylate anion in 7.24), 7.03 (d, J = 8.3 Hz, 2H), 6.95 (t, J = 8.0 Hz,
1H), 4.79 (s, 2H), 4.17 (s, 2H), 3.80 (dt, J = 13 Hz, J = 5 Hz, 2H),
3.49−3.70 (m, 8H), 3.33−3.49 (bm, 8H), 3.06−3.30 (bm, 8H), 2.88−
3.06 (bm, 4H), 2.82 (dt, J = 14 Hz, J = 6 Hz, 2H), 2.35 (s, 3H; tosylate
anion), 2.15−2.18 (m, 2H), 2.00 (d, J = 15 Hz, 2H), 1.79 (m, 2H),
1.66 (m, 2H), 1.25−1.45 (m, 16H); 13C NMR (100.6 MHz, CD3OD,
25 °C) δppm 199.9, 174.8, 174.5, 168.9, 167.3 (d, J = 254 Hz), 162.8 (q,
J = 34 Hz; TFA), 143.3 (tosylate anion), 142.9 (tosylate anion), 142.1,
134.2, 132.1 (d, J = 9.5 Hz), 130.6, 130.0 (tosylate anion), 126.8
(tosylate anion), 121.8, 118.2, 117.7 (q, J = 291 Hz; TFA), 116.8 (d,
J = 22 Hz), 64.8, 60.1, 57.6, 56.1, 55.9, 53.6, 53.0, 51.1, 50.4, 49.7, 49.3,
42.1, 36.0, 30.39, 30.36, 30.36, 30.1, 30.0, 28.5, 28.0, 27.6, 27.5,
24.7, 21.4 (tosylate anion); 19F NMR (376.3 MHz, CD3OD, 25 °C)
δppm −78.6, −108.6; HRMS (TOF MS ES negative) m/z calcd for
C48H71N7O8F− [M − H]− 892.5348, found 892.5347.
Compound 5b was obtained as a very viscous clear liquid; yield
1.24 g, (2.25 mmol, 89% yield); 1H NMR (400 MHz, CDCl3, 25 °C)
δppm 7.25−7.29 (m, 2H), 6.83−6.98 (m, 7H), 4.66 (s, 2H), 4.00 (t, J =
6.3 Hz, 2H), 3.61 (t, J = 6.6 Hz, 2H), 3.41 (t, J = 7.2 Hz, 2H), 2.59−
2.89 (bm, 8H), 1.97−2.01 (m, 2H), 1.53−1.68 (bm, 6H), 1.27−1.32
(bm, 14H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 174.3, 157.1
(d, J = 238 Hz), 155.1, 142.9, 129.2, 118.7, 115.6 (d, J = 23 Hz), 115.4
(d, J = 8 Hz), 115.1, 66.9, 63.3, 62.7, 60.6, 54.9, 49.6, 40.7, 32.7,
29.4, 29.32, 29.32, 29.27, 29.27, 29.1, 27.3, 26.8, 26.6, 25.7; 19F NMR
(376.3 MHz, CDCl3, 25 °C) δppm −124.7; FAB+-MS m/z 554.3
[M + H]+.
Preparation of 6a and 6b. A solution of 5a (0.97 g, 1.81 mmol,
1 equiv) or 5b (1.00 g, 1.81 mmol, 1 equiv) with dry triethylamine
(TEA; 1.46 g, 2.0 mL, 14.5 mmol, 8 equiv) in dry DCM (45 mL) was
cooled to 0 °C, and then tosyl chloride (0.69 g, 3.62 mmol, 2 equiv)
and a catalytic amount of 4-dimethylaminopyridine (DMAP) were
added. The solution was stirred for about 3 h (until TLC indicated the
disappearance of 5a or 5b) at 10 °C. Water (70 mL) was added, and
the organic layer was separated. After extraction with DCM (3 ×
70 mL), the combined organic phase was dried over magnesium sul-
fate, filtered, and evaporated under reduced pressure, and the residue
was purified by column chromatography over silica. The eluting
solvent was exchanged from chloroform to 2% methanol in chloroform
(Rf of the product was 0.1) to afford 6a as a very viscous clear liquid;
1
yield 1.04 g (1.45 mmol, 80%); H NMR (400 MHz, CDCl3, 25 °C)
δppm 8.00−8.04 (m, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.1 Hz,
2H), 7.22−7.27 (m, 2H), 7.12 (t, J = 8.6 Hz, 2H), 6.81−6.90 (m, 3H),
4.65 (s, 2H), 4.00 (t, J = 6.5 Hz, 2H), 3.40 (t, J = 7.3 Hz, 2H), 3.01 (t,
J = 7.2 Hz, 2H), 2.83 (bs, 4H), 2.63 (bm, 2H), 2.49−2.53 (m, 2H),
2.44 (s, 3H), 1.96 (t, J = 7.0 Hz, 2H), 1.58−1.65 (bm, 6H), 1.10−1.31
(bm, 14H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 198.6, 174.3,
165.6 (d, J = 254 Hz), 144.6, 143.0, 133.6, 133.2, 131.7 (d, J = 9 Hz),
129.8, 129.2, 127.8, 118.8, 115.6 (d, J = 22 Hz), 115.2, 70.7, 63.4, 60.6,
57.6, 49.6, 40.8, 36.4, 29.36, 29.31, 29.27, 29.11, 28.85, 28.77, 27.6,
27.3, 26.7, 25.3, 21.9, 21.6; 19F NMR (376.3 MHz, CDCl3, 25 °C)
δppm −106.9; FAB+-MS m/z 720.3 [M + H]+.
Compound 6b was obtained as a very viscous clear liquid; yield
0.95 g (1.34 mmol, 74%); 1H (400 MHz, CDCl3, 25 °C) δppm 7.78 (d,
J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 7.24−7.29 (m, 2H), 6.83−
6.98 (bm, 7H), 4.67 (s, 2H), 4.01 (t, J = 6.4 Hz, 4H), 3.38−3.43 (m,
Compound 9b was obtained nearly quantitatively as a light brown
1
viscous substance; H NMR (400 MHz, CD3OD, 25 °C) δppm 7.71
7008
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(d, J = 8.1 Hz, 2H; tosylate anion), 7.28 (t, J = 7.9 Hz, 2H), 7.23 (d, J =
8.0 Hz, 2H; tosylate anion), 6.96−7.14 (m, 4H), 6.88−6.95 (m, 3H),
4.78 (s, 2H), 4.21 (s, 2H), 4.05 (t, J = 5.7 Hz, 2H), 3.84 (dt, J = 13 Hz,
J = 5 Hz, 2H), 3.49−3.70 (m, 8H), 3.33−3.49 (m, 10H), 3.16−3.24
(m, 2H), 2.88−3.16 (m, 6H), 2.83 (dt, J = 11 Hz, J = 5 Hz, 2H), 2.36
(s, 3H; tosylate anion), 2.15−2.30 (m, 2H), 1.99 (d, J = 15 Hz, 2H),
1.73−1.86 (m, 2H), 1.60−1.71 (m, 2H), 1.25−1.46 (m, 16H); 13C
NMR (100.6 MHz, CD3OD, 25 °C) δppm 174.8, 174.3, 168.7, 162.2
(q, J = 36 Hz; TFA), 158.9 (d, J = 234 Hz), 156.1, 143.58, 143.53
(tosylate anione), 141.8 (tosylate anione), 130.5 (tosylate anione),
129.9, 127.0 (tosylate anione), 121.6, 118.2, 117.8 (q, J = 291 Hz; TFA),
116.8 (d, J = 23 Hz), 116.8 (d, J = 8 Hz), 66.5, 64.8, 60.0, 56.1,
56.0, 55.9, 53.4, 53.1, 51.2, 50.8, 42.1, 30.57, 30.58, 30.58, 30.59, 30.33,
30.27, 28.7, 28.2, 27.9, 27.7, 25.4, 24.7, 21.3 (tosylate anione); 19F
NMR (376.3 MHz, CD3OD, 25 °C) δppm −77.5, −126.2; HRMS
(TOF MS ES negative) m/z calcd for C47H71N7O8F− [M − H]−
880.5348, found 880.5345.
Preparation of 10a and 10b. A concentrated methanolic solu-
tion of 9a (220 mg, 0.15 mmol) or 9b (220 mg, 0.15 mmol) was
mixed with an aqueous solution of gadolinium(III) chloride hexa-
hydrate (155 mg, 0.15 mmol). The pH of the obtained solution was
adjusted to 7.8, and the solution was stirred at room temperature (rt)
for 1 week, during which the pH was measured and readjusted to 7.8.
After lyophilization 10a was obtained as a brown solid. The reaction
proceeded nearly quantitatively; HRMS (TOF MS ES positive) m/z
calcd for C48H70N7O8F155Gd+ [M + H]+ 1046.4496, found 1046.4506.
Compound 10b was obtained nearly quantitatively as a brown solid;
HRMS (TOF MS ES positive) m/z calcd for C47H70N7O8F155Gd+
[M + H]+ 1034.4496, found 1034.4496.
was purified by column chromatography over silica. The eluting
solvent was changed from chloroform to 3% methanol in chloroform,
to afford 12a as a very viscous colorless liquid; yield 0.46 g (0.87 mmol,
1
69%); H NMR (200 MHz, CDCl3, 25 °C) δppm 7.94−8.10 (m, 2H),
7.22−7.38 (m, 3H), 7.13 (t, J = 8.6 Hz, 1H), 6.81−6.95 (m, 3H), 4.79
(s, 2H), 3.47−3.80 (m, 12H), 3.03 (t, J = 7.0 Hz, 2H), 2.75−2.90 (m,
4H), 2.35−2.75 (m, 4H), 1.98 (q, J = 7.0 Hz, 2H), 1.60−1.75 (m,
2H); 13C NMR (50.3 MHz, CDCl3, 25 °C) δppm 198.5, 174.6, 165.6
(d, J = 254 Hz), 143.1, 133.5, 130.6 (d, J = 9 Hz), 129.1, 119.1, 115.8,
115.5 (d, J = 21 Hz), 72.4, 70.3, 69.2, 64.7, 61.6, 60.4, 57.5, 49.5, 40.9,
36.3, 29.6, 29.1, 21.7; 19F NMR (188.15 MHz, CDCl3, 25 °C)
δppm −106.2; FAB+-MS m/z 528.1 [M + H]+.
Compound 12b was obtained as a very viscous colorless liquid;
1
yield 0.55 g (0.97 mmol, 76%); H NMR (400 MHz, CDCl3, 25 °C)
δppm 7.90−7.99 (m, 2H), 7.18 (t, J = 7.5 Hz, 2H), 7.06 (t, J = 8.2 Hz,
2H), 6.87 (d, J = 8.1 Hz, 2H), 6.77 (t, J = 7.2 Hz, 1H), 4.72 (s, 2H),
3.34−3.75 (m, 16H), 2.70−3.05 (m, 6H), 2.55−2.70 (m, 2H), 2.45−
2.55 (m, 2H), 1.87−2.00 (m, 2H), 1.56−1.68 (m, 2H); 13C NMR
(100.6 MHz, CDCl3, 25 °C) δppm 198.1, 174.1, 165.3 (d, J = 254 Hz),
142.8, 133.2, 130.4 (d, J = 9 Hz), 128.9, 118.7, 115.4 (d, J = 22 Hz),
115.3, 72.3, 70.3, 70.2, 70.03, 69.95, 68.9, 64.5, 61.2, 59.9, 57.2, 49.2,
40.7, 36.0, 28.6, 21.1; 19F NMR (376.3 MHz, CDCl3, 25 °C)
δppm −106.0; FAB+-MS m/z 572.3 [M + H]+.
Compound 12c was obtained as a very viscous colorless liquid; yield
1
0. (0.94 mmol, 74%); H NMR (400 MHz, CDCl3, 25 °C) δppm 7.27
(t, J = 8 Hz, 2H), 6.94 (t, J = 8 Hz, 4H), 6.80−6.90 (m, 3H), 4.79 (s,
2H), 4.00 (t, J = 6.3 Hz, 2H), 3.52−3.75 (m, 12H), 2.75−2.95 (m,
4H), 2.55−2.75 (m, 4H), 1.93−2.05 (m, 2H), 1.63−1.74 (m, 2H); 13C
NMR (100.6 MHz, CDCl3, 25 °C) δppm 174.7, 157.1 (d, J = 238 Hz),
155.1, 143.1, 129.2, 119.1, 115.7, 115.7 (d, J = 23 Hz), 115.4 (d, J = 8
Hz), 72.5, 70.4, 70.4, 69.1, 66.9, 64.7, 61.6, 60.4, 54.9, 49.6, 41.0, 29.2,
26.9; 19F NMR (376.3 MHz, CDCl3, 25 °C) δppm −124.7; FAB+-MS
m/z 516.2 [M + H]+.
Preparation of 11a and 11b. Solution of tosyl chloride (TsCl;
6.3 g, 33.4 mmol, 1 equiv) in dry DCM (25 mL) at rt was dropwise
(during 0.5 h) added into a cooled (0 °C) solution of triEG (25.0 g,
167 mmol, 5 equiv) or tetraEG (32.4 g, 167 mmol, 5 equiv) and dry
TEA (9.2 mL, 6.7 g, 67 mmol, 2 equiv) in dry DCM (100 mL). The
resulting mixture was stirred at rt for 1.5 h. After washing with water,
extracting with chloroform, drying with magnesium sulfate, filtering,
and evaporation, a colorless viscous liquid was obtained. It was purified
by column chromatography over silica. The eluting solvent was
exchanged from chloroform to 5% methanol in chloroform (Rf of the
monotosylated product 11a was 0.35; Rf of the ditosylated product was
0.8; triEG stayed near the start line; the TLC was exposed by solution
of potassium permanganate) to afford 11a as a clear, colorless viscous
Compound 12d was obtained as a very viscous colorless liquid;
1
yield 0.50 g (0.90 mmol, 71%); H NMR (400 MHz, CDCl3, 25 °C)
δppm 7.26 (t, J = 7.6 Hz, 2H), 6.91−6.99 (m, 4H), 6.81−6.89 (m, 3H),
4.78 (s, 2H), 4.00 (t, J = 6.2 Hz, 2H), 3.57−3.73 (m, 14H), 3.53 (t, J =
4.3 Hz, 2H), 2.80−2.93 (m, 4H), 2.57−2.71 (m, 4H), 1.93−2.04 (m,
2H), 1.64−1.73 (m, 2H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm
174.5, 157.0 (d, J = 238 Hz), 155.0, 143.0, 129.0, 118.9, 115.6, 115.5
(d, J = 23 Hz), 115.3 (d, J = 8 Hz), 72.4, 70.4, 70.3, 70.1, 70.0, 69.0,
66.8, 64.6, 61.4, 60.3, 54.8, 49.5, 40.8, 29.0, 26.7; 19F NMR (376.3
MHz, CDCl3, 25 °C) δppm −124.7; FAB+-MS m/z 560.3 [M + H]+.
Preparation of 13a, 13b, 13c, and 13d. TsCl (0.14 g, 0.76
mmol, 2 equiv) and a catalytic amount of DMAP were added to a
cooled (10 °C) solution of 12a (0.2 0g, 0.38 mmol, 1 equiv), 12b
(0.22 g, 0.38 mmol, 1 equiv), 12c (0.20 g, 0.38 mmol, 1 equiv), or 12d
(0.21 g, 0.38 mmol, 1 equiv) and TEA (0.3 mL, 2.3 mmol, 6 equiv) in
dry DCM (10 mL). After about 3 h of stirring at rt, water (20 mL) and
DCM (15 mL) were added. The organic layer was separated, and after
extraction with dichloromethane (2 × 25 mL) the combined organic
phase was dried over magnesium sulfate, filtered, and evaporated
under reduced pressure. The residue was purified by column chroma-
tography over silica. The eluting solvent was changed from chloroform
to 3% methanol in chloroform to afford 13a as a viscous liquid; yield
1
liquid; yield 7.2g, (23.7 mmol; 71%); H NMR (400 MHz, CDCl3,
25 °C) δppm 7.79 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 4.16 (t,
J = 4.7 Hz, 2H), 3.69 (t, J = 4.6 Hz, 4H), 3.53−3.61 (m, 6H), 2.44 (s,
3H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 144.6, 132.5, 129.5,
127.5, 72.2, 70.3, 69.8, 69.0, 68.2, 61.2, 21.2; FAB+-MS m/z 305.1
[M + H]+, 327.1 [M + Na]+.
Compound 11b was purified by column chromatography over silica.
The eluting solvent was exchanged from chloroform to 5% methanol
in chloroform (Rf of the monotosylated product was 0.4; Rf of the
ditosylated product was 0.75; Rf of 11b was 0.1, when TLC of 11b was
exposed in the manner of 11a) to afford 11b as a clear, colorless
1
viscous liquid; yield 7.2 g (20.7 mmol, 62%); H NMR (400 MHz,
CDCl3, 25 °C) δppm 7.76 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H),
4.12 (t, J = 4.7 Hz, 2H), 3.35−3.80 (m, 14H), 2.41 (s, 3H); 13C NMR
(100.6 MHz, CDCl3, 25 °C) δppm 144.7, 132.8, 129.7, 127.8, 72.3,
70.6, 70.5, 70.3, 70.2, 69.2, 68.5, 61.5, 21.5; FAB+-MS m/z 349.0
[M + H]+, 371.0 [M + Na]+, 387.0 [M + K]+.
Preparation of 12a, 12b, 12c, and 12d. Compound 11a (0.50 g,
1.65 mmol, 1.3 equiv) or 11b (0.57 g, 1.65 mmol, 1.3 equiv) was
added under argon to a solution of 3a (0.50 g, 1.27 mmol, 1 equiv) or
3b (0.49 g, 1.27 mmol, 1 equiv) and KOH (0.092 g, 1.65 mmol, 1.3
equiv) in dry DMF (15 mL), and the mixture was heated at 110 °C
for 20 h. Water (60 mL) and chloroform (50 mL) were added, and the
organic layer was separated. After extraction with chloroform (3 ×
60 mL), the combined organic phase was dried over magnesium
sulfate, filtered, and evaporated under reduced pressure (first, the
chloroform was evaporated in an evaporator, and then the DMF in
high vacuum system with liquid nitrogen). The obtained viscous liquid
1
0.21 g (0.31 mmol, 82%); H NMR (400 MHz, CDCl3, 25 °C) δppm
7.98−8.06 (m, 2H), 7.77 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H),
7.23 (t, J = 8.3 Hz, 2H), 7.13 (t, J = 8.6 Hz, 2H), 6.82−6.90 (m, 3H),
4.75 (s, 2H), 4.11 (t, J = 4.8 Hz, 2H), 3.67 (t, J = 4.8 Hz, 4H), 3.56−
3.62 (m, 6H), 3.02 (t, J = 7.1 Hz, 2H), 2.75−2.90 (m, 4H), 2.45−2.65
(m, 4H), 2.44 (s, 3H), 1.97 (q, J = 6.5 Hz, 2H), 1.61−1.70 (m, 2H);
13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 198.5, 174.6, 165.6 (d, J =
254 Hz), 144.8, 144.8, 143.1, 132.9, 130.7 (d, J = 9 Hz), 129.8, 129.2,
127.9, 118.9, 115.6, 115.5 (d, J = 22 Hz), 70.7, 70.7, 70.3, 69.4, 69.1,
68.7, 64.7, 57.6, 49.6, 40.9, 36.4, 29.7, 29.2, 21.6; 19F NMR (376.3 MHz,
CDCl3, 25 °C) δppm −106.2; FAB+-MS m/z 682.1 [M + H]+.
Compound 13b was obtained as a viscous liquid; yield 0.22 g
1
(0.30 mmol, 79%); H NMR (400 MHz, CDCl3, 25 °C) δppm 7.97−
8.05 (m, 2H), 7.77 (d, J = 7.9 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.23
(t, J = 7.7 Hz, 2H), 7.12 (t, J = 8.3 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H),
7009
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6.83 (t, J = 7.2 Hz, 1H), 4.77 (s, 2H), 4.09 (t, J = 4.7 Hz, 2H), 3.48−
3.71 (m, 14H), 3.03 (t, J = 6.9 Hz, 2H), 2.75−2.99 (m, 4H), 2.45−
2.75 (m, 4H), 2.43 (s, 3H), 2.00 (q, J = 6.8 Hz, 2H), 1.62−1.71 (m,
2H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 198.5, 174.6, 165.6
(d, J = 254 Hz), 144.7, 143.1, 130.6 (d, J = 9 Hz), 129.8, 129.8, 129.2,
127.9, 118.9, 115.6 (d, J = 22 Hz), 115.6, 115.5, 70.6, 70.5, 70.4, 70.3,
69.2, 69.1, 68.6, 64.7, 60.2, 57.4, 49.5, 41.0, 36.3, 29.6, 29.0, 21.6; 19F
NMR (376.3 MHz, CDCl3, 25 °C) δppm −106.1; FAB+-MS m/z 726.3
[M + H]+.
Compound 13c was obtained as a viscous liquid; yield 0.22 g
(0.32 mmol, 85%); 1H NMR (400 MHz, CDCl3, 25 °C) δppm 7.76 (d,
J = 8.3 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.25 (t, J = 7.4 Hz, 2H), 6.95
(d, J = 8.3 Hz, 2H), 6.91 (d, J = 8.1 Hz, 2H), 6.81−6.88 (m, 3H), 4.76
(s, 2H), 4.11 (t, J = 4.7 Hz, 2H), 4.01 (t, J = 6.3 Hz, 2H), 3.64−3.69
(m, 4H), 3.56−3.62 (m, 6H), 2.75−2.95 (m, 4H), 2.55−2.75 (m, 4H),
2.43 (s, 3H), 1.95−2.05 (m, 2H), 1.64−1.71 (m, 2H); 13C NMR
(100.6 MHz, CDCl3, 25 °C) δppm 174.5, 157.1 (d, J = 238 Hz), 155.1,
144.8, 143.0, 132.9, 129.8, 129.1, 127.8, 118.9, 115.6 (d, J = 23 Hz),
115.5, 115.4 (d, J = 8 Hz), 70.7, 70.2, 69.2, 69.1, 68.7, 66.9, 64.6, 60.3,
54.9, 49.6, 40.9, 29.2, 26.8, 21.6; 19F NMR (376.3 MHz, CDCl3,
25 °C) δppm −124.7; FAB+-MS m/z 670.1 [M + H]+.
Compound 13d was obtained as a viscous liquid; yield 0.21 g (0.29
mmol, 77%); 1H NMR (400 MHz, CDCl3, 25 °C) δppm 7.78 (d,
J = 8.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.24 (t, J = 7.5 Hz, 2H), 6.95
(d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H), 6.80−6.87 (m, 3H), 4.78
(s, 2H), 4.10 (t, J = 4.8 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.40−3.75
(m, 16H), 2.75−2.95 (m, 2H), 2.55−2.75 (m, 2H), 2.44 (s, 3H),
1.90−2.10 (m, 2H), 1.60−1.85 (m, 4H); 13C NMR (100.6 MHz,
CDCl3, 25 °C) δppm 174.5, 157.1 (d, J = 238 Hz), 155.1, 144.8, 143.1,
133.0, 129.8, 129.2, 127.9, 118.9, 115.7 (d, J = 23 Hz), 115.6, 115.5 (d,
J = 8 Hz), 70.7, 70.6, 70.6, 70.5, 70.4, 69.3, 69.2, 68.7, 67.0, 64.7, 60.3,
55.0, 49.7, 49.7, 41.1, 29.7, 29.3, 21.6; 19F NMR (376.3 MHz, CDCl3,
25 °C) δppm −124.8; FAB+-MS m/z 714.3 [M + H]+.
5.2 Hz, 2H), 3.47 (s, 2H), 2.10−3.40 (bm, 27H; includes s of tosylate
anione (0.75H) in 2.31), 2.03 (q, J = 6.8 Hz, 2H), 1.62−1.70 (m, 2H),
1.37−1.55 (m, 31H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm
174.4, 172.9, 172.6, 157.1 (d, J = 238 Hz), 155.0, 144.3, 142.9, 138.4,
129.2, 128.2, 126.2, 119.2, 115.7 (d, J = 23 Hz), 115.6, 115.4 (d, J = 8
Hz), 82.2, 82.1, 70.1, 70.0, 69.0, 68.0, 66.8, 64.5, 60.2, 56.3, 56.3, 55.6,
54.9, 52.4, 50.5, 49.7, 49.5, 49.5, 40.9, 29.1, 28.1, 28.0, 27.8, 21.2; 19F
NMR (376.3 MHz, CDCl3, 25 °C) δppm −124.7; FAB+-MS m/z
1034.3 [M + Na]+.
Compound 14d was obtained as a viscous liquid; yield 0.13 g
(0.12 mmol, 81%); 1H NMR (400 MHz, CDCl3, 25 °C) δppm 7.81 (d,
J = 8.1 Hz, 2H; tosylate anione), 7.22 (t, J = 7.8 Hz, 2H), 7.10 (d, J =
8.0 Hz, 2H; tosylate anione), 6.90−6.99 (m, 4H), 6.78−6.86 (m, 3H),
4.79 (s, 2H), 4.01 (t, J = 6.1 Hz, 2H), 3.70 (t, J = 4.9 Hz, 2H),
3.53−3.65 (m, 10H), 3.49 (t, J = 5.2 Hz, 2H), 1.90−3.40 (bm, 27H;
includes s of tosylate anione (3H) in 2.31), 1.71 (bs, 12H), 1.36−1.53
(m, 27H); 13C NMR (100.6 MHz, CDCl3, 25 °C) δppm 173.0, 172.6,
172.6, 157.2 (d, J = 238 Hz), 154.9, 144.0, 142.8, 138.7, 129.4, 128.4,
126.2, 119.1, 115.8 (d, J = 23 Hz), 115.4 (d, J = 8 Hz), 115.4, 82.1,
82.0, 77.2, 70.4, 70.4, 70.2, 70.0, 69.9, 69.0, 67.7, 64.6, 56.4, 56.4, 55.7,
55.0, 52.3, 49.7, 49.5, 41.0, 28.6, 28.0, 27.9, 27.9, 21.3; 19F NMR
(376.3 MHz, CDCl3, 25 °C) δppm −124.5; FAB+-MS m/z 1078.5
[M + Na]+.
Preparation of 15a, 15b, 15c, and 15d. Compound 14a (0.10 g,
0.10 mmol, 1 equiv), 14b (0.11 g, 0.10 mmol, 1 equiv), 14c (0.10 g,
0.10 mmol, 1 equiv), or 14d (0.11 g, 0.10 mmol, 1 equiv) was dis-
solved in TFA (1.5 g, 1 mL, 8.8 mmol, 88 equiv). After stirring at
ambient temperature for 4 h the solution was evaporated in reduced
pressure. Methanol was added and evaporated, and then chloroform
was added and evaporated (until the complete disappearance of TFA)
1
to afford 15a nearly quantitatively as a grayish viscous substance; H
NMR (400 MHz, CD3OD, 25 °C) δppm 7.90−8.05 (m, 2H), 7.60 (d,
J = 7.5 Hz, tosylate anion), 7.19 (t, J = 7.4 Hz, 2H), 7.12 (d, J =
7.5 Hz, tosylate anion), 7.10−7.16 (m, 2H), 6.91 (d, J = 7.8 Hz, 2H),
6.75−6.85 (m, 1H), 4.75 (s, 2H), 4.07 (bs, 4H), 2.60−3.80 (m, 38H),
2.24 (s, tosylate anion), 1.94−2.10 (m, 4H), 1.82−1.94 (m, 2H); 13C
NMR (100.6 MHz, CD3OD, 25 °C) δppm 198.7, 174.5, 174.2, 168.9,
167.3 (d, J = 254 Hz), 162.8 (q, J = 36 Hz; TFA), 143.7 (tosylate
anion), 143.5 (tosylate anion), 141.9, 134.4, 132.0 (d, J = 9 Hz), 130.6,
130.0 (tosylate anion), 126.9 (tosylate anion), 121.4, 117.7, 117.6 (q,
J = 291 Hz; TFA), 116.6 (d, J = 22 Hz), 71.7, 71.0, 70.8, 69.1, 65.4,
59.8, 57.6, 55.6, 53.5, 52.8, 52.3, 50.4, 49.9, 42.0, 36.0, 28.5, 21.3
(tosylate anion), 19.5; 19F NMR (376.3 MHz, CD3OD, 25 °C)
δppm −78.6, −109.1; HRMS (MALDI-TOF) m/z calcd for C43H63N7O10F+
[M + H]+ 856.4620, found 856.4556; m/z calcd for C43H62N7O10FNa+
[M + Na]+ 878.4440, found 878.4473.
Preparation of 14a, 14b, 14c, and 14d. Compound 7 (0.08 g,
0.15 mmol, 1 equiv) was added to a stirred solution of 13a (0.10 g,
0.15 mmol, 1 equiv), 13b (0.11 g, 0.15 mmol, 1 equiv), 13c (0.10 g,
0.15 mmol, 1 equiv), or 13d (0.11 g, 0.15 mmol, 1 equiv) and K2CO3
(0.10 g, 0.75 mmol, 5eq.) in ACN (4 mL). After heating at 80 °C for
3.5 to 4 h the precipitate was filtered, and the filtrate was evaporated
under reduced pressure. The residue was purified by column chroma-
tography over silica. The eluting solvent was changed from chloroform
to 20% methanol in chloroform to afford 14a as a viscous liquid; yield
0.107 g (0.105 mmol, 70%); 1H NMR (400 MHz, CDCl3, 25 °C) δppm
7.90−8.10 (m, 2H), 7.70−7.90 (m, 2H), 7.21 (t, J = 7.8 Hz, 2H),
7.03−7.16 (m, 4H), 6.87 (d, J = 8.2 Hz, 2H), 6.83 (t, J = 7.8 Hz, 1H),
4.73 (s, 2H), 3.44−3.70 (m, 8H), 1.8−3.3 (m, 41H; includes s of
tosylate anione in 2.29), 1.61−1.70 (m, 2H), 1.30−1.60 (m, 27H); 13C
NMR (100.6 MHz, CDCl3, 25 °C) δppm 198.5, 174.2, 173.1, 172.7,
165.6 (d, J = 254 Hz), 144.3, 142.8, 138.6, 132.9, 130.7 (d, J = 9 Hz),
129.3, 128.3, 126.2, 119.2, 115.6 (d, J = 22 Hz), 115.6, 82.1, 82.0, 70.1,
70.0, 69.0, 68.1, 64.6, 57.3, 56.3, 55.7, 52.4, 50.7, 50.5, 50.4, 50.1, 49.7,
49.4, 40.9, 36.2, 29.6, 28.9, 28.0, 27.9, 21.2; 19F NMR (376.3 MHz,
CDCl3, 25 °C) δppm −106.1; FAB+-MS m/z 1046.5 [M + Na]+.
Compound 14b was obtained as a viscous liquid; yield 0.12 g
Compound 15b was was obtained nearly quantitatively as a grayish
1
viscous substance; H NMR (400 MHz, CD3OD, 25 °C) δppm 8.01−
8.08 (m, 2H), 7.68 (d, J = 8.0 Hz, tosylate anion), 7.28 (t, J = 7.8 Hz,
2H), 7.21 (d, J = 7.8 Hz, tosylate anion), 7.15−7.19 (m, 2H), 7.00 (d,
J = 8.1 Hz, 2H), 6.90 (t, J = 7.2 Hz, 1H), 4.85 (s, 2H), 4.15 (bs, 2H),
2.80−3.90 (m, 46H), 2.33 (s, tosylate anion), 2.05−2.26 (m, 2H),
1.95−2.05 (m, 2H); 13C NMR (100.6 MHz, CD3OD, 25 °C) δppm
198.7, 174.5, 174.2, 168.9, 167.0 (d, J = 254 Hz), 160.9 (q, J = 36 Hz;
TFA), 143.8 (tosylate anion), 143.5 (tosylate anion), 141.8, 134.4,
132.0 (d, J = 9 Hz), 130.7, 129.9 (tosylate anion), 127.0 (tosylate
anion), 121.4, 117.8, 117.2 (q, J = 291 Hz; TFA), 116.7 (d, J = 22 Hz),
71.7, 71.4, 71.3, 71.2, 71.1, 69.2, 65.6, 59.8, 57.7, 55.6, 53.5, 52.8, 52.3,
50.5, 49.9, 49.8, 42.1, 36.0, 28.5, 28.3, 21.3 (tosylate anion), 19.5; 19F
NMR (376.3 MHz, CD3OD, 25 °C) δppm −78.5, −109.0; HRMS
(TOF MS ES negative) m/z calcd for C45H65N7O11F− [M − H]−
898.4726, found 898.4725.
1
(0.11 mmol, 73%); H NMR (400 MHz, CDCl3, 25 °C) δppm 7.95−
8.03 (m, 2H), 7.80 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 7.8 Hz, 2H), 7.05−
7.15 (m, 4H), 6.89 (d, J = 8.2 Hz, 2H), 6.82 (t, J = 7.8 Hz, 1H), 4.75
(s, 2H), 3.43−3.69 (m, 18H), 1.90−3.35 (m, 35H; includes s of
tosylate anione in 2.29), 1.63−1.73 (d, 2H), 1.30−1.60 (m, 27H); 13C
NMR (100.6 MHz, CDCl3, 25 °C) δppm 198.4, 174.1, 173.0, 172.6,
165.5 (d, J = 254 Hz), 144.2, 142.7, 138.7, 133.3, 130.7 (d, J = 9 Hz),
129.3, 128.3, 126.2, 119.1, 115.6 (d, J = 22 Hz), 115.5, 82.1, 82.0, 77.2,
70.3, 70.1, 70.0, 69.8, 68.9, 67.6, 64.6, 56.3, 55.6, 52.2, 50.6, 50.6, 49.7,
49.4, 40.9, 36.1, 29.6, 28.0, 28.0, 27.8, 27.8, 21.2, 21.2; 19F NMR (376.3
MHz, CDCl3, 25 °C) δppm −106.1; FAB+-MS m/z 1090.3 [M + Na]+.
Compound 14c was obtained as a viscous liquid; yield 0.11 g
(0.11 mmol, 73%); 1H NMR (400 MHz, CDCl3, 25 °C) δppm 7.82 (d,
J = 8.1 Hz, 0.5H; tosylate anione), 7.24−7.29 (m, 2H), 7.09 (d, J = 8.1 Hz,
0.5H; tosylate anione), 6.89−7.02 (m, 4H), 6.81−6.89 (m, 3H),
4.75 (s, 2H), 4.02 (t, J = 6.2 Hz, 2H), 3.57−3.67 (m, 8H), 3.55 (t, J =
Compound 15c was was obtained nearly quantitatively as a grayish
viscous substance; 1H NMR (400 MHz, CD3OD, 25 °C) δppm 7.68 (d,
J = 8.0 Hz, 1H; tosylate anion), 7.28 (t, J = 7.4 Hz, 2H), 7.21 (d, J =
7.9 Hz, 1H; tosylate anion), 6.93−7.02 (m, 4H), 6.80−6.93 (m, 3H),
4.78 (s, 2H), 3.95−4.17 (m, 4H), 2.60−3.90 (m, 38H), 2.34 (s, 1.5H;
tosylate anion), 2.05−2.31 (m, 4H), 1.95−2.04 (m, 2H); 13C NMR
(100.6 MHz, CD3OD, 25 °C) δppm 174.6, 174.2, 169.1, 162.4 (q, J =
36 Hz; TFA), 158.9 (d, J = 237 Hz), 156.1, 143.7, 143.5 (tosylate anion),
7010
dx.doi.org/10.1021/jo400646k | J. Org. Chem. 2013, 78, 7001−7012

The Journal of Organic Chemistry
Article
141.9 (tosylate anion), 130.8 (tosylate anion), 129.9, 127.0 (tosylate
anion), 121.8, 118.3, 117.9 (q, J = 291 Hz; TFA), 116.8 (d, J = 23 Hz),
116.8 (d, J = 8 Hz), 71.8, 71.0, 69.2, 66.5, 65.6, 59.8, 55.8, 55.3, 53.6,
52.3, 50.6 42.1, 28.7, 25.4, 21.3 (tosylate anion); 19F NMR (376.3
MHz, CDCl3, 25 °C) δppm −77.5, −126.2; HRMS (TOF MS ES
negative) m/z calcd for C42H61N7O10F− [M − H]− 842.4464, found
842.4463.
Compound 15d was was obtained nearly quantitatively as a grayish
viscous substance; 1H NMR (400 MHz, CD3OD, 2 5 °C) δppm 7.71 (d,
J = 8.0 Hz, 1H; tosylate anion), 7.26−7.36 (m, 2H), 7.23 (d, J = 7.9 Hz,
1H; tosylate anion), 6.96−7.06 (m, 4H), 6.87−6.96 (m, 3H), 4.81
(s, 2H), 4.07 (bs, 4H), 2.70−3.95 (m, 44H), 2.36 (s, 1.5H; tosylate
anion), 2.13−2.35 (bs, 2H), 1.95−2.13 (m, 2H); 13C NMR (100.6
MHz, CD3OD, 25 °C) δppm 174.6, 173.9, 169.1, 162.9 (q, J = 35 Hz;
TFA), 158.9 (d, J = 238 Hz), 156.1, 143.8, 143.6 (tosylate anion),
141.8 (tosylate anion), 130.6 (tosylate anion), 129.9, 127.0 (tosylate
anion), 119.6, 118.1, 117.9 (q, J = 291 Hz; TFA), 116.8 (d, J = 23 Hz),
116.8 (d, J = 8 Hz), 71.6, 71.4, 71.3, 71.1, 69.1, 68.9, 66.5, 65.6, 60.1,
55.8, 55.1, 54.0, 52.5, 52.3, 42.1, 40.2, 33.1, 30.7, 30.5, 25.4, 23.7, 21.3
(tosylate anion); 19F NMR (376.3 MHz, CDCl3, 25 °C) δppm −77.4,
−126.1; HRMS (TOF MS ES negative) m/z calcd for C44H65N7O11F−
[M − H]− 886.4726, found 886.4722.
Preparation of 16a, 16b, 16c, and 16d. A concentrated aqueous
solution of 15a, 15b, 15c, or 15d (0.15 mmol of each one) was mixed
with an equimolar aqueous solution of gadolinium(III) chloride hexa-
hydrate (155 mg, 0.15 mmol). The pH of the obtained solution was
adjusted to 7.8, and the solution was stirred at rt for 1 week, during
which the pH was measured and readjusted to 7.8. After lyophiliza-
tion 16a was obtained as a grayish solid. The reaction proceeded
nearly quantitatively; HRMS (TOF MS ES negative) m/z calcd for
C43H58N7O10F158Gd− [M − H]− 1009.3470, found 1009.3481.
Compound 16b was obtained nearly quantitatively as a brown solid;
HRMS (TOF MS ES positive) m/z calcd for C45H64N7O11F158Gd+
[M + H]+ 1055.3889, found 1055.3893.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial suppport from the Office of the Chief Scientist of the
Ministry of Health of the State of Israel (Grant No. 6225) is
gratefully acknowledged. We thank professor Moshe Rehavi
and Ms. Netta Roz from the Department of Physiology and
Pharmacology of Tel Aviv University for the binding affinity
tests with our target-specific CAs. We thank Dr. Noam Tal and
Mr. Shimon Hauptmann from School of Chemistry of Tel Aviv
University for the HRMS measurements.
REFERENCES
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Compound 16c was obtained nearly quantitatively as a brown solid;
HRMS (TOF MS ES positive) m/z calcd for C42H60N7O10F155Gd+
[M + H]+ 996.3612, found 996.3615.
Compound 16d was obtained nearly quantitatively as a brown solid;
HRMS (TOF MS ES positive) m/z calcd for C44H64N7O11F155Gd+
[M + H]+ 1040.3874, found 1040.3868.
Dopamine receptor binding was evaluated according to known
protocol.21 Striatal tissues was isolated from C57 bl. mouse and
homogenized in 100 vol of ice-cold Tris-HCl 50 mM pH 7.4 buffer,
using Brinkman Polytron. The homogenate was centrifuged three
times (and resuspended twice in equal volumes of buffer) for 20 min
at 3000 rpm. Final reconstitution of the pellet was done to yield a
tissue concentration of 10 mg wt weight per mL buffer. To analyze the
dopamine receptor binding, 0.1 mL of [3H]-spiperone (5 × 10−10 M)
was added to 0.1 mL of striatal membranes and 0.8 mL Tris-HCl
buffer and incubated at 25 °C. One hour later, the mixture was diluted
in 3 mL of ice-cold buffer and filtered under vacuum through glass
fiber filters (Whatman GF/C). The filters were washed three times
with 3 mL of ice-cold buffer. The bound radioactivity was counted in a
liquid scintillation cocktail (Optiflour) using a scintillation counter
(Tri carb 300c Packard). The specific binding was defined as the
difference between the binding in the presence and absence of 1 μM
sulpiride.
ASSOCIATED CONTENT
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L.; Yang, H.; Grossniklaus, H. E.; Liu, Z.-R.; Yang, J. J. PlosOne 2011,
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S
* Supporting Information
NMR spectra, MS or HRMS of all compounds prepared, as
well as HRMS of the gadolinium complexes. This material is
available free of charge via the Internet at http://pubs.acs.org.
(13) (a) Molecular Neurobiology of the Mammalian Brain, 2nd ed.;
McGeer, P. L., Eccles, J. C., McGeer, E. G., Eds.; Plenum Press: New
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ycohen@post.tau.ac.il.
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