Synthesis and characterization of an MRI Gd-based probe designed to target the translocator protein

Article abstract of DOI:10.1002/mrc.3919

DPA-713 is the lead compound of a recently reported pyrazolo[1,5-a] pyrimidineacetamide series, targeting the translocator protein (TSPO 18 kDa), and as such, this structure, as well as closely related derivatives, have been already successfully used as p

Full text of DOI:10.1002/mrc.3919

Research Article
Received: 19 July 2012
Revised: 12 September 2012
Accepted: 11 December 2012
Published online in Wiley Online Library: 10 January 2013
(wileyonlinelibrary.com) DOI 10.1002/mrc.3919
Synthesis and characterization of an MRI
Gd-based probe designed to target the
translocator protein
Erika Cerutti,a Annelaure Damont,b Frédéric Dollé,b Simona Baronic and
Silvio Aimea*
DPA-713 is the lead compound of a recently reported pyrazolo[1,5-a]pyrimidineacetamide series, targeting the translocator
protein (TSPO 18 kDa), and as such, this structure, as well as closely related derivatives, have been already successfully used
as positron emission tomography radioligands. On the basis of the pharmacological core of this ligands series, a new magnetic
resonance imaging probe, coded DPA-C6-(Gd)DOTAMA was designed and successfully synthesized in six steps and 13% overall
yield from DPA-713. The Gd-DOTA monoamide cage (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) repre-
sents the magnetic resonance imaging reporter, which is spaced from the phenylpyrazolo[1,5-a]pyrimidineacetamide moiety
(DPA-713 motif) by a six carbon-atom chain. DPA-C6-(Gd)DOTAMA relaxometric characterization showed the typical behavior
of a small-sized molecule (relaxivity value: 6.02 mMꢀ1 sꢀ1 at 20 MHz). The good hydrophilicity of the metal chelate makes DPA-
C6-(Gd)DOTAMA soluble in water, affecting thus its biodistribution with respect to the parent lipophilic DPA-713 molecule. For
this reason, it was deemed of interest to load the probe to a large carrier in order to increase its residence lifetime in blood.
Whereas DPA-C6-(Gd)DOTAMA binds to serum albumin with a low affinity constant, it can be entrapped into liposomes (both
in the membrane and in the inner aqueous cavity). The stability of the supramolecular adduct formed by the Gd-complex and
liposomes was assessed by a competition test with albumin. Copyright © 2013 John Wiley & Sons, Ltd.
1
Keywords: NMR; H; MRI; contrast agents; relaxometry; Gd-complexes; DPA-713; TSPO
a resolution of 100mm is attained), but its major drawback is repre-
Introduction
sented by the low sensitivity of its probes.[28]
The translocator protein (TSPO-18 kDa), formerly known as
peripheral benzodiazepine receptor,[1] is a five-transmembrane
protein[2] localized primarily on the outer mitochondrial mem-
brane.[1,3] TSPO is expressed in many organs, but it is expressed
at the highest level in steroidogenic cells, where TSPO is involved
in the translocation of cholesterol from the outer to the inner
mitochondrial membranes. TSPO is also located in the central
nervous system where it is usually expressed in microglia[4,5]
and in some neuronal cell types.[6–9] Following neuronal injury,
TSPO expression is dramatically increased, in particular, in the
case of Alzheimer disease and multiple sclerosis.[10,11] Microglia
are extremely sensitive toward alterations due to neuronal injury.
Until now, positron emission tomography has been the most
used medical imaging technique in the study of how microglia
are involved in degenerative diseases. Different TSPO targeting
molecules have been synthesized over the past two decades.
They belong to different chemical classes,[12–14] but the most
promising one is the 2-phenylpyrazolo[1,5-a]pyrimidineacetamide
series[15,16], which includes, among others, the two following radio-
tracers: [11C]DPA-713[17–19] and [18 F]DPA-714.20–25,27,28
A novel MRI Gd-based probe has therefore been designed,
combining the pharmacological properties of the DPA-713 core
with a gadolinium-dedicated (DOTA) chelator. The conjugation
site was selected on the basis of previous SAR studies having
shown that chemical flexibility was permitted at the para position
of the phenyl ring.[29] A spacer of six carbon atoms was moreover
introduced to minimize the steric hindrance of the DOTA cage at
the interaction site of the molecule with TSPO. As the develop-
ment of an MRI targeting agent requires systems that remain in
circulation for a long time, the interaction of DPA-C6-(Gd)
DOTAMA with human serum albumin (HSA) and its incorporation
into liposomes have been explored. Moreover, the latter systems
can act as carriers for a great number of imaging reporter units
thus offering a route to overcome the sensitivity issue associated
to MR applications.
*
Correspondence to: Silvio Aime, Dipartimento di Chimica and Centro di
Imaging Molecolare, Università degli Studi di Torino, V. Nizza 52, 10126 Torino,
Italy. E-mail: silvio.aime@unito.it
Magnetic resonance imaging (MRI) is a non-invasive technique
that allows morphology, physiology and metabolism visualization
in vivo. MRI images deal with water 1H proton spatial localization,
whose intensity is sufficiently high, thanks to the high concentra-
tion of water in living organisms. The main advantage of this tech-
nique is its superb spatial resolution (in ordinary image acquisition,
a Dipartimento di Chimica and Centro di Imaging Molecolare, Università degli
Studi di Torino, Via Nizza 52, 10126, Torino, Italy
b CEA, I2BM, Service Hospitalier Frédéric Joliot, 4 Place du Général Leclerc,
F-91406, Orsay, France
c
Invento Srl, Via Nizza 52, Torino, Italy
Magn. Reson. Chem. 2013, 51, 116–122
Copyright © 2013 John Wiley & Sons, Ltd.

Gd-probe targeting TSPO
Results and Discussion
DPA-C6-(Gd)DOTAMA (1) was synthesized in six steps from
DPA-713 and obtained in 13% overall and non-optimized yield
(Scheme 1). Briefly, DPA-713, as starting material, was synthe-
sized according to literature procedures.[17] O-demethylation of
DPA-713 was carried out with a 1 M solution of BBr3 in dichloro-
methane at low temperature (from ꢀ60 to ꢀ20 ꢁC) affording the
corresponding phenol 2 (DPA-OH) in high yield (95%).[20] The
aminohexyl spacer was introduced into the structure by coupling
the DPA-skeleton 2 with 6-(Boc-amino)hexan-1-ol activated as a
sulfonate (synthesized as described in the experimental part) in
DMSO at 50 ꢁC in the presence of NaOH as a base, to afford product
3 in moderate yield (63%). The Boc protecting group was removed
using trifluoroacetic acid in dichloromethane, giving the free amine
(4) in 80% yield. Coupling of 4 with DOTA(tBu)3[30] was carried out
in the presence of TBTU and DIPEA in DMF yielding the DOTAMA
derivative 5 in 80% yield. Tert-Butyl groups were then removed
using trifluoroacetic acid to afford 6 (DPA-C6-DOTAMA) in 33%
yield after chromatography purification. The ligand complexation
was carried out by stoichiometric addition of GdCl3 maintaining
the pH at 6.5 by addition of a 0.1N aqueous NaOH solution.
Figure 1. 1/T1 nuclear magnetic resonance dispersion profile of 1 mM
aqueous solution of DPA-C6-(Gd)DOTAMA (■) and human serum albumin
adduct (□) at 25 ꢁC and pH = 7.4.
Table 1. Best fitting parameters for from the analysis of the nuclear
magnetic resonance dispersion profile of 1
Relaxometric characterization of DPA-C6-(Gd)DOTAMA (1)
Δ2
tv
tr tm
q
r1p
(1019 sꢀ2
)
(ps) (ps) (ms)
(mMꢀ1 sꢀ1 a
)
The relaxivity of 1 (i.e. the change of the water proton relaxation
rate in the presence of the paramagnetic complex at 1 mM
concentration) measured at 20 MHz and 20 ꢁC was 6.02 mMꢀ1 sꢀ1
.
DPA-C6-(Gd)DOTAMA (1)
2.65
1.4
27 114 1.4
40 70 1.3
1
1
6.02
4.7
This value was slightly higher than that reported in literature[31] for
the parent Gd-DOTAMA complex (r1p = 4.7 mMꢀ1 sꢀ1). The nuclear
magnetic resonance dispersion (NMRD) profile was also acquired
(Figure 1) and found to show the typical behavior of small
gadolinium complexes.
Gd-DOTAMA-C6-OH
aMeasured at 25 ꢁC at 20 MHz.
The NMRD data were fitted to the values calculated on the
basis of the established paramagnetic relaxation theory,[32] and
the obtained values are reported in Table 1 together with the
corresponding ones of the parent Gd-DOTAMA-C6-OH. Going
from Gd-DOTAMA-C6-OH to 1, the increased molecular weight
is responsible for the markedly longer molecular re-orientational
time tr, which in turn is the main determinant of the increase of
the observed relaxivity.
Interaction of DPA-C6-(Gd)DOTAMA (1) with HSA
Human serum albumin is the most abundant protein in blood
and often acts as carrier for metabolites, nutrients and drugs.
The lipophilicity of the DPA-713 moiety anticipates the possible
binding of 1 to HSA. Binding parameters (the affinity constant KA
and the relaxivity of supramolecular adduct r1pbound) were deter-
mined using the proton relaxation enhancement (PRE) method.[33]
The experimental procedure consists of carrying out a titration in
which a fixed quantity of Gd-complex (0.19 mM) is titrated with
increasing concentrations of the macromolecular host (Figure 2).
The observed relaxation rates increase according to KA and r1bound
values. In this case, a KA of 1100Mꢀ1 and an r1bound of 20mMꢀ1 sꢀ1
were determined.
The obtained KA value demonstrates that there is a relatively
bound
weak interaction between 1 and albumin. The calculated r1
is markedly higher than the r1p of the free DPA-C6-(Gd)DOTAMA
(1), in accordance with the formation of the adduct with albumin
that is characterized by a markedly slower tumbling rate.
The NMRD profile of a solution containing DPA-C6-(Gd)
DOTAMA (1) and HSA in ca. 1:1 ratio (0.19 mM of 1 and 0.2 mM
of HSA) was acquired (data not shown). On the basis of the pre-
viously mentioned reported results, compound 1 is only in part
bound to HSA. From the knowledge of the affinity constant (KA)
and the millimolar relaxivity of the bound fraction (r1bound), it
Scheme 1. Synthesis of DPA-C6-(Gd)DOTAMA. (i) BBr3, CH2Cl2 anhydr.,
ꢀ60 ꢁC to ꢀ20 ꢁC, 5 h; (ii) 6-((tert-butoxycarbonyl) amino)hexyl 4-methyl-
benzenesulfonate, NaOH, DMSO, 50 ꢁC, overnight; (iii) TFA, CH2Cl2, RT,
24 h; (iv) DOTA(tBu)3, TBTU, DIPEA, DMF, RT, 24 h; (v) TFA, CH2Cl2, RT,
24 h; (vi) GdCl3 ꢂ 6H2O, pH = 6.5, RT.
Magn. Reson. Chem. 2013, 51, 116–122
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

E. Cerutti et al.
pursue a slow release of the compound of interest. Two types of
liposome have been prepared, namely LP1 and LP2. Both lipo-
somes were prepared by means of the thin film hydration
method.[34] The latter procedure consists of two key-steps, namely
a first step that leads to the lipidic film that is hydratated (second
step) with a saline buffer solution (HEPES/NaCl mole ratio 1/6.75).
Vesicles were formulated by using 72% (for LP1) or 87% (for
LP2) of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine),
10% of cholesterol and 3% of DSPE-PEG-methoxy-2000
(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethyleneglycol)-2000] (ammonium salt)). For LP1, 15% of 1
was mixed with the lipidic components to yield the lipidic film. In
LP2, 1 was dissolved in the hydration solution (20 mM). By the latter
procedure, DPA-C6-(Gd)DOTAMA is encapsulated in the aqueous
cavity of the liposome. The mean hydrodynamic diameters of the
liposomes were determined by dynamic light scattering. They were
found to be very similar thus showing that the methodology of
preparation is not as relevant as the formulation in determining
the final size of the vesicles (Table 3).
Figure 2. Determination of binding association constants KA between
DPA-C6-(Gd)DOTAMA (1) and human serum albumin at 25 ꢁC and pH = 7.4.
The relaxivity values, measured at 25 ꢁC at 20 MHz, were found
to be 9.69 and 7.85 mMꢀ1 sꢀ1 for LP1 and LP2, respectively. These
values are higher than the value obtained for free 1, as conse-
quence of the interaction with the liposome membrane. Overall,
the observed relaxation enhancement is lower than the one
usually observed for amphiphilic Gd complexes interacting with
a liposomial membrane. For both types of liposomes, one may
think that an equilibrium is occurring in the inner cavity for which
DPA-C6-(Gd)DOTAMA distributes between the inner membrane
layer and the aqueous solution. Thus, the NMRD profiles of both
liposomes were analyzed taking into account either the contribu-
tion due to 1 ‘free’ (in the inner cavity) and the one due to 1
incorporated in the membrane. Equation (1) has been used:
has been possible to quantify as 68% the percentage of com-
pound 1 bound to HSA under the applied 1:1 ratio. From these
data, the NMRD profile for the fraction of DPA-C6-(Gd)DOTAMA
bound to HSA has been calculated (Figure 1), and the high field
relaxivity values fitted according to the theory of paramagnetic
relaxation enhancement. The values obtained for the relevant
parameters are reported in Table 2.
On going from the free complex (1) to the adduct with HSA, a
high relaxivity peak centered at ca. 40 MHz is observed for the
latter species indicative of a relaxivity domain determined by
the molecular re-orientational time (tr). In fact, from the NMRD
analysis, tr is markedly increased if compared with free DPA-C6-
(Gd)DOTAMA, as expected. The relaxivity obtained at 20 MHz
for the macromolecular adduct with HSA is 17.5 mMꢀ1 sꢀ1, i.e.
almost three times larger than the value obtained for free DPA-
C6-(Gd)DOTAMA.
free
bound
r1p ¼ Xfreeꢃr1p þ Xboundꢃr1p
(1)
where Xbound and Xfree are the molar fraction of 1 that is bound or
free
not bound to the liposome’s membrane, r1p
is the relaxivity
value of 1 (6.02 mMꢀ1 sꢀ1) and r1p
is the relaxivity value of
bound
the adduct DPA-C6-(Gd)DOTAMA-liposome. We fixed the latter
value to 16 mMꢀ1 sꢀ1, that is the relaxivity value for analogous
Gd-DOTAMA system incorporated in liposome’s membrane pre-
viously investigated in our laboratory.[35] 1/T1 NMRD profiles for
both liposome suspensions were acquired in the high field
region, namely from 20 to 70 MHz, as it is known that for para-
magnetic macromolecular systems, this is the field strength
where the acquired T1 data are less prone to yield erroneous
results in the fitting procedure. On the basis of Xfree and Xbound
Entrapment of DPA-C6-(Gd)DOTAMA (1) in liposomes
Entrapment into liposomes of DPA-C6-(Gd)DOTAMA (1) has been
taken into account to increase the circulation lifetime and to
Table 2. Best fitting parameters for DPA-C6-(Gd)DOTAMA/HSA
Δ2 (1019 sꢀ2
)
tv (ps) tr (ps)
24 59 000
t
m (ms)
q
1
DPA-C6-(Gd)DOTAMA/HSA
1.15
1.1
bound
values (obtained from KA) the NMRD profiles of the r1p
Table 3. Liposome formulations and main parameters
Membrane
Molar ratio
Cavity
Dimension (nm)
151
r1p (mMꢀ1 sꢀ1 a
)
LP1
LP2
POPC
Chol
72%
10%
3%
Saline buffer
9.69
DSPE-PEG-metoxy 2000
DPA-C6-(Gd)DOTAMA
POPC
15%
87%
10%
3%
DPA-C6-(Gd)DOTAMA 20 mM
149
7.85
Chol
DSPE-PEG-metoxy 2000
aMeasured at 25 ꢁC at 20 MHz.
wileyonlinelibrary.com/journal/mrc
Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2013, 51, 116–122

Gd-probe targeting TSPO
values for the two types of liposomes have been extracted
(Figure 3).
Conclusion
The 2-phenylpyrazolo[1,5-a]pyrimidineacetamide DPA-713 was
successfully derivatized and coupled to the DOTA cage. The
resulting ligand was complexed with gadolinium. The relaxivity
of the obtained Gd-complex was slightly higher (6.02 mMꢀ1 sꢀ1
at 20 MHz) than the previously reported ones for analogous Gd-
DOTAMA complexes, which is in accordance with its increased
molecular weight. The NMRD profile is consistent with the one
expected for a small, fast tumbling gadolinium complex. Interac-
tion with HSA was assessed and a KA of 1100 M-1 was calculated.
Incorporation into liposomes displayed a relaxivity ‘hump’ in the
NMRD profile, as a demonstration of the formation of a supramo-
lecular adduct. Interestingly, it has been found that the amphi-
philic DPA-C6-(Gd)DOTAMA complex can be either incorporated
in the liposomial membrane and in the inner aqueous cavity.
Overall, the availability of liposomes loaded with this complex is
promising for future in vivo studies with increased half-life in
blood stream and encourages pursuing the task of the slow
release of MRI targeting agents.
The calculated NMRD profiles of the bound fraction were then
fitted as earlier recalled,[32] and the best fitting values for the rel-
evant parameters are reported in Table 4.
The ‘humps’ at high magnetic field observed for both
liposomes are an indication that part of the Gd complex is inter-
acting with the liposomial membrane. As discussed earlier, the
detection of a relaxivity enhancement at 30–40 MHz is an indi-
cation of the occurrence of an increased tr with respect to the
value found for the free complex. Also, the exchange lifetime
of the coordinated water molecule (tm) appears slightly in-
creased upon entrapment of the Gd complex in the liposome.
This behavior may be accounted for in terms of a slight distor-
tion in the co-ordination cage geometry upon the binding of
the lipophilic moiety to the liposome’s membrane.
The relaxivity of DPA-C6-(Gd)DOTAMA (1) loaded liposomes
was then measured in the presence of increasing amounts of
albumin (HSA) to evaluate whether some amount of 1 could
transfer from the liposome to HSA. The Gd-loaded liposomes
were challenged with increasing concentrations of albumin (from
0.1 to 0.8 mM), but the relaxation rate (R1oss) values remained
unchanged. Although the relaxivity of 1 bound to HSA is similar
to that of 1 incorporated in the liposome’s membrane, one would
expect that the release of 1 from liposomes to HSA causes an
overall shift of concentration of 1 either in the aqueous cavity or
in the phospholipide bilayer. Such a shift would be related to the
amount of added HSA, and it would result in a change of the
observed water proton relaxation rate. The observed constancy
of R1 clearly indicates that the Gd-complex is not leaving the
liposomes in the presence of HSA in the suspending medium. This
result is encouraging in the light of future in vivo experiments as
DPA-C6-(Gd)DOTAMA loaded liposomes appear to be a stable
carrier for improving the blood lifetime of this imaging agent.
Experimental Section
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-
ethylene glycol)-2000] (ammonium salt) (DSPE-PEG-methoxy-2000)
were purchased from Avanti Polar Inc. (Alabaster, Al, USA);
DOTA(tBu)3 was purchased from CheMatech, and all the other
chemicals were purchased from Sigma-Aldrich and used with
no further purification. Solvents were stored over molecular
sieves and purged with argon before use. Column chromatogra-
phies were conducted on silica gel (0.63–0.200 mm, VWR). Thin
layer chromatography (TLC) was carried out using aluminum
pre-coated plates of silica gel 60 F254 (VWR). Compounds were
detected using a UV-lamp (254 nm) and by sprinkling the TLC
plate with KMnO4 aqueous solution or ninhydrin/ethanol solu-
tion and heating.
1H-NMR and 13C-NMR spectra for characterization of synthe-
sized products were recorded on a Bruker (Wissembourg, France)
Avance (400 MHz) apparatus or on a Bruker Avance600 apparatus
using the hydrogenated residue of the deuterated solvents CDCl3
(d = 7.26 ppm), CD2Cl2 (d = 5.32 ppm) or CD3OD (d = 3.31 ppm) for
1H-NMR as well as the deuterated solvents CDCl3 (d = 77.2 ppm),
CD2Cl2 (d = 54.0 ppm) or CD3OD (d = 49.0 ppm) as an internal
standard for 13C-NMR. Chemical shifts are reported in parts per
million (ppm) downfield from TMS. Multiplicities are reported as
s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet)
and b (broad). The mass spectra (MS) were recorded on a Thermo
Electron Ion Trap LCQ Deca XP + spectrometer (positive electro-
spray ionization (ESI+)) or on a Waters 3100 Single quadrupole
mass detector.
The longitudinal water proton relaxation rate was measured by
using a Stelar Spinmaster (Stelar, Mede, Pavia, Italy) spectrometer
operating at 20 MHz by means of the standard inversion recovery
sequence. The temperature was controlled with a Stelar VTC-91
air-flow heater equipped with a copper constantan thermocouple
(uncertainty of 0.1ꢁC). The proton 1/T1 NMRD profiles were
measured over a continuum of magnetic field strength from
0.00024 to 0.47 T (corresponding to 0.0120 MHz proton Larmor
frequency) on a Stelar field-cycling relaxometer. The relaxometer
works under complete computer control with an absolute
Figure 3. 1/T1 nuclear magnetic resonance dispersion profile of 1 mM
aqueous solution of LP1 (♦) and LP2 (▲) at 25 ꢁC and pH = 7.4.
Table 4. Best fitting parameters for liposomes LP1 and LP2
Δ2 (1019 s-2)
tv (ps)
tr (ps)
tm (ms)
q
LP1
LP2
1.56
1.19
0.15
7.06
3000
2500
0.88
0.87
1
1
Magn. Reson. Chem. 2013, 51, 116–122
Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

E. Cerutti et al.
uncertainty in 1/T1 ofꢄ 1%. Data points from 0.47 T (20 MHz) to
1.7T (70 MHz) were collected on a Stelar Spinmaster spectrometer
working at variable field.
Liposomes extrusion was carried out with Lipex extruder,
Northern Lipids Inc., Canada and the liposomes size measurements
were performed on Zetasizer NanoZS Dynamic Light Scattering
(Malvern, UK).
[CH2, CH2NHBoc], 30.0 [CH2], 28.8 [CH2], 28.5 [3ꢂ CH3, t-Bu], 26.2
[CH2], 25.2 [CH2], 21.7 [CH3, PhCH3].
Step 2. DPA-OH (2) (1.17 g, 3.33 mmol) was dissolved in 50 ml of
DMSO, then NaOH (0.23 g, 6.66 mmol) and 6-((tert-butoxycarbo-
nyl)amino)hexyl 4-methylbenzenesulfonate (1.47 g, 4.00 mmol)
were added. The solution was stirred at 50 ꢁC overnight. The reac-
tion was poured onto a saturated aqueous solution of K2CO3
(150 ml) and extracted twice with EtOAc (2 ꢂ 200 ml). The com-
bined organic layers were washed twice (2 ꢂ 200 ml) with brine,
dried over Na2SO4, filtered and concentrated under reduced
pressure. The crude was purified on silica gel using CH2Cl2/
CH3OH 98/2 v/v as eluent to afford the desired product 3
(1.07 g, 61%) as a yellow solid. Rf (CH2Cl2/CH3OH 95/5 v/v): 0.33.
1H-NMR (CDCl3, 400 MHz) d 7.75 (2H, d, J = 8.8 Hz, Ar), 6.96 (2H,
d, J = 8.8 Hz, Ar), 6.50 (1H, s, Ar), 3.99 (2H, t, J = 6.4 Hz, ArOCH2),
3.91 (2H, s, CH2C = O), 3.50 (2H, q, J = 7.2 Hz, NCH2CH3), 3.41 (2H,
q, J = 7.2 Hz, NCH2CH3), 3.13 (2H, m, CH2NHBoc), 2.73 (3H, s,
ArCH3), 2.53 (3H, s, ArCH3), 1.80 (2H, m, OCH2CH2), 1.53–1.39
(6H, m, 3 ꢂ CH2), 1.44 (9H, s, t-Bu), 1.20 (3H, t, J = 7.2 Hz, NCH2CH3),
1.11 (3H, t, J = 7.2 Hz, NCH2CH3). 13C-NMR (CDCl3, 100 MHz) d
170.2 [C, C = O], 159.1 [C, Ar], 157.6 [C, Ar], 156.2 [C, C = O],
155.2 [C, Ar], 147.8 [C, Ar], 144.8 [C, Ar], 130.0 [2 ꢂ CH, Ar], 126.3
[C, Ar], 114.6 [2 ꢂ CH, Ar], 108.2 [CH, Ar], 100.9 [C, Ar], 79.2 [C, C
(CH3)3], 67.9 [CH2, ArOCH2], 42.4 [CH2, NCH2CH3], 40.7 [CH2,
NCH2CH3], 30.2 [CH2, CH2NHBoc], 29.3 [CH2], 28.5 [3 ꢂ CH3, t-
Bu], 28.3 [CH2], 26.7 [CH2], 25.9 [2 ꢂ CH2], 24.8 [CH3, ArCH3], 17.1
[CH3, ArCH3], 14.5 [CH3, NCH2CH3], 13.2 [CH3, NCH2CH3]. MS ESI +
(m/z) 553 [M + H]+, 574 [M + Na]+.
Chemistry
N,N-diethyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]
pyrimidin-3-yl)acetamide (DPA-713). Synthesized according to
Ref. 17.
Rf (CH2Cl2/CH3OH 95/5 v/v): 0.39. 1H-NMR (CD2Cl2, 400 MHz)
d 7.75 (2H, d, J = 8.8 Hz, Ar), 6.99 (2H, d, J = 8.8 Hz, Ar), 6.54 (1H,
s, Ar), 3.88 (2H, s, CH2CO), 3.85 (3H, s, OCH3), 3.51 (2H, q,
J = 7.2 Hz, NCH2CH3), 3.39 (2H, q, J = 7.2 Hz, NCH2CH3), 2.72 (3H,
s, ArCH3), 2.53 (3H, s, ArCH3), 1.22 (3H, t, J = 7.2 Hz, NCH2CH3),
1.11 (3H, t, J = 7.2 Hz, NCH2CH3). 13C-NMR (CD2Cl2 100 MHz)
d 170.3 [C, CO], 160.4 [C, Ar], 158.1 [C, Ar], 155.0 [C, Ar], 148.3
[C, Ar], 145.3 [C, Ar], 130.2 [2 ꢂ CH, Ar], 127.0 [C, Ar], 114.4 [2 ꢂ CH,
Ar], 108.7 [CH, Ar], 101.3 [C, Ar], 55.8 [CH3, OCH3], 42.8 [CH2,
NCH2CH3], 41.0 [CH2, NCH2CH3], 28.6 [CH2, COCH2], 24.9
[CH3, ArCH3], 17.1 [CH3, ArCH3], 14.7 [CH3, NCH2CH3], 13.4 [CH3,
NCH2CH3]. MS ESI + (m/z) 367 [M + H]+.
N,N-diethyl-2-(2-(4-hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]
pyrimidin-3-yl)acetamide,(DPA-OH, 2). Synthesized according to
Ref 20.
1
Rf (CH2Cl2/CH3OH 92/8 v/v): 0.41. H-NMR (CD2Cl2, 400 MHz) d
2-(2-(4-((6-aminohexyl)oxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimi-
din-3-yl)-N,N-diethylacetamide (4). Compound 3 (1.80 g, 3.26 mmol)
was dissolved in 50 ml of dichloromethane and trifluoroacetic acid
(10 ml, 132 mmol) was added. The solution was stirred at room
temperature overnight. The reaction was then evaporated to
dryness, and the residue was partitioned between a saturated
aqueous solution of K2CO3 and EtOAc. The organic layer was col-
lected, and the aqueous phase was washed twice with EtOAc.
The resulting organic layers were combined to the first one.
The combined organic layers were washed with brine, dried over
Na2SO4, filtered and concentrated under reduced pressure. The
crude was purified on silica gel (CH2Cl2/CH3OH 90/10) to afford
the desired product (1.47 g, 80%) as an oil. Rf (CH2Cl2/CH3OH
80/20 v/v): 0.25. 1H-NMR (CD3OD, 400 MHz) d 7.62 (2H, d,
J = 8.4 Hz, Ar), 7.13 (1H, s, Ar), 7.06 (2H, d, J = 8.4 Hz, Ar), 4.07
(4H, m, ArOCH2, CH2CO), 3.56 (2H, q, J = 7.2 Hz, NCH2CH3), 3.44
(2H, q, J = 7.2 Hz, NCH2CH3), 2.95 (5H, m, CH2NH2 + ArCH3), 2.77
(3H, s, ArCH3), 1.86 (2H, m, OCH2CH2), 1.71 (2H, m, CH2NH2),
1.60–1.50 (4H, m, 2 ꢂ CH2), 1.27 (3H, t, J = 7.2 Hz, NCH2CH3) 1.16
(3H, t, J = 7.2 Hz, NCH2CH3). 13C-NMR (CD3OD, 100 MHz) d 171.0
[C, C = O], 161.9 [C, Ar], 159.8 [C, Ar], 159.0 [C, Ar], 155.5 [C, Ar],
141.5 [C, Ar], 131.2 [2 ꢂ CH, Ar], 125.0 [C, Ar], 115.9 [2 ꢂ CH, Ar],
109.3 [CH, Ar], 100.0 [C, Ar], 69.0 [CH2, ArOCH2], 43.8 [CH2,
NCH2CH3], 42.2 [CH2, NCH2CH3], 40.7 [CH2, CH2NH2], 30.1 [CH2],
28.8 [CH2], 28.5 [CH2], 27.2 [CH2], 26.7 [CH2], 21.1 [CH3, ArCH3],
17.9 [CH3, ArCH3], 14.3 [CH3, NCH2CH3], 13.3 [CH3, NCH2CH3].
MS ESI + (m/z) 452 [M + H]+.
7.56 (2H, d, J = 8.4 Hz, Ar), 6.82 (2H, d, J = 8.4 Hz, Ar), 6.55 (1H, s,
Ar), 3.90 (2H, s, CH2CO), 3.47 (2H, q, J = 7.2 Hz, NCH2CH3), 3.37
(2H, q, J = 7.2 Hz, NCH2CH3), 2.71 (3H, s, ArCH3), 2.52 (3H, s, ArCH3),
2.21 (1H, bs, OH), 1.18 (3H, t, J = 7.2 Hz, NCH2CH3), 1.09 (3H, t,
J = 7.2 Hz, NCH2CH3). 13C-NMR (CD2Cl2 100 MHz) d 171.2 [C, CO],
158.7 [C, Ar], 158.2 [C, Ar], 155.8 [C, Ar], 148.2 [C, Ar], 145.8[C, Ar],
130.4 [2 ꢂ CH, Ar], 125.4 [C, Ar], 116.0 [2 ꢂ CH, Ar], 108.9 [CH, Ar],
100.8 [C, Ar], 43.0 [CH2, NCH2CH3], 41.4 [CH2, NCH2CH3], 28.7
[CH2, COCH2], 24.7 [CH3, ArCH3], 17.2 [CH3, ArCH3], 14.4 [CH3,
NCH2CH3], 13.3 [CH3, NCH2CH3]. MS ESI + (m/z) 353. M + H]+.
tert-Butyl (6-(4-(3-(2-(diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo
[1,5-a]pyrimidin-2-yl)phenoxy)hexyl)carbamate (3). Step 1. 6-((tert-
butoxycarbonyl)amino)hexyl 4-methylbenzenesulfonate) synthesis.
Under argon atmosphere, 6-(Boc-amino)1-hexanol (1 g, 4.6 mmol)
was dissolved in 25 ml of dichloromethane. Triethylamine (1.28 ml,
9.2 mmol), para-toluenesulfonyl chloride (1.05 g, 5.52 mmol) and a
catalytic amount of DMAP were added. The solution was stirred at
room temperature overnight. After this time, the solvent was
evaporated, and the residue was partitioned between ethyl
acetate (150 ml) and HCl 0.1 M aqueous solution (150 ml). The
organic layer was separated, and the aqueous phase was
extracted with EtOAc (2 ꢂ 150 ml); the combined organic layers
were washed once with brine (150 ml), dried over Na2SO4,
filtered and concentrated under reduced pressure. The
crude was purified on silica gel (heptane/acetone 80/20) to afford
6-((tert-butoxycarbonyl)amino)hexyl
4-methylbenzenesulfonate
(1.47 g, 83%) as a colorless oil. Rf (heptane/EtOAc 1/1v/v): 0.50.
1H-NMR (CDCl3, 400 MHz) d 7.77 (2H, d, J = 8.0 Hz, Ph), 7.34
(2H, d, J = 8 Hz, Ph), 4.00 (2H, t, J = 6.4 Hz TsOCH2), 3.05 (2H, m,
CH2CH2NHBoc), 2.44 (3H, s, PhCH3), 1.63 (2H, m, TsOCH2CH2),
1.42 (9H, s, t-Bu), 1.39–1.24 (6H, m, 3 ꢂ CH2). 13C-NMR (CDCl3
100 MHz) d 156.1 [C, CO], 144.8 [C, Ph], 133.3 [C, Ph], 130 [2ꢂ CH,
Ph], 128.0 [2ꢂ CH, Ph], 79.2 [C, C(CH3)3], 70.6 [CH2, TsOCH2], 40.5
2,20,200-(10-(2-((6-(4-(3-(2-(diethylamino)-2-oxoethyl)-5,7-dimethylpyra-
zolo[1,5-a]pyrimidin-2-yl)phenoxy)hexyl)amino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid (5)[30]
.
DOTA(tBu)3
(0,500 g, 0.87 mmol) was dissolved in 25 ml of DMF. TBTU
(0,28 g, 0.87 mmol), DIPEA (1.72 ml, 6.70 mmol) and compound
4 (0,327 g, 0,67 mmol) were added. The solution was stirred at
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Copyright © 2013 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2013, 51, 116–122

Gd-probe targeting TSPO
room temperature for 24 h. The reaction was partitioned between
water and EtOAc. The aqueous phase was acidified to pH 5–6,
and extracted twice with EtOAc. The collected organic layers were
washed once with water and once with brine, dried over Na2SO4,
filtered and evaporated under reduced pressure. The residue was
purified on silica gel using CH2Cl2/CH3OH 95/5v/v as eluent, yield-
ing the desired product 5 as a yellow solid (0.55 g, 80%). Rf (CH2Cl2/
0.12 mmol), maintaining the pH value at 6.5 with NaOH 0.1N.
The mixture was allowed to stir at RT until the pH remained con-
stant at 6.5. The solution was then lyophilized to give a pale yellow
solid (150mg, impurities are only salts). The amount of residual
free Gd3+ ion was assessed by the orange xylenol UV method,[36]
and the corresponding amount of ligand was added to avoid
the presence of free Gd ions. MS ESI + (m/z) 992 [M + H]+, 496
[(M + 2H)/2]+.
1
CH3OH 90/10 v/v): 0.22. H-NMR (CD3OD, 400 MHz) d 7.64 (2H, d,
J = 8.8Hz, Ar), 7.00 (2H, d, J = 8.8Hz, Ar), 6.79 (1H, s, Ar), 4.03 (2H, t,
J = 6.0Hz, ArOCH2), 3.96 (2H, s, CH2C = O), 3.60–2.50 (8H, b, 4 ꢂ
NCH2COOtBu), 3.56 (2H, q, J = 7.2Hz, NCH2CH3), 3.41 (2H, q,
J = 7.2Hz, NCH2CH3), 3.22 (2H, t, J = 6.8Hz, CH2NH-DOTA(tBu)3),
2.75 (3H, s, ArCH3), 2.56 (3H, s, ArCH3), 2.50–2.00 (16H, b, 8 ꢂ CH2)
1.81 (2H, m, OCH2CH2), 1.60–1.30 (6H, m, 3 ꢂ CH2), 1.49 (27H, s,
3 ꢂ t-Bu), 1.24 (3H, t, J = 7.2Hz, NCH2CH3), 1.12 (3H, t, J = 7.2 Hz,
NCH2CH3). 13C-NMR (CD3OD, 100 MHz) d 174.4 [2ꢂ C, C = O],
174.1 [C, C = O], 173.1 [C, C = O], 172.3 [C, C = O], 161.1 [C, Ar],
160.0 [C, Ar], 156.5 [C, Ar], 148.9 [C, Ar], 147.0 [C, Ar], 131.0 [2ꢂ CH,
Ar], 126.9 [C, Ar], 115.5 [2ꢂ CH, Ar], 109.7 [CH, Ar], 101.5 [C, Ar], 82.8
[3ꢂ C, C(CH3)3], 68.9 [CH2, ArOCH2], 57.2 [CH2, NCH2CON], 56.8
[CH2, NCH2COOtBu], 56.7 [2ꢂ CH2, NCH2COOtBu], 55–51 [8 ꢂ CH2,
b, CH2], 43.6 [CH2, NCH2CH3], 42.0 [CH2, NCH2CH3], 40.2 [CH2], 30.8
[CH2], 30.3 [CH2], 28.9 [CH2], 28.5 [9ꢂ CH3, 3 ꢂ t-Bu], 27.8 [CH2],
27.0 [CH2], 24.2 [CH3, ArCH3], 16.9 [CH3, ArCH3], 14.3 [CH3,
NCH2CH3], 13.3 [CH3, NCH2CH3]. MS ESI + (m/z) 1028 [M+ Na]+.
Liposomes preparation, general procedure
Large unilamellar vescicles (LUV) were prepared following the
thin film hydration method.[34] Briefly, the lipids were dissolved
in chloroform or chloroform/methanol solution, and the organic
solution was slowly evaporated until the thin film was formed.
The film was then hydrated at 55 ꢁC with 1.45 ml of saline buffer
(HEPES/NaCl mole ratio 1/6.75) to obtain a total lipid amount of
30 mg/ml. The resulting suspension of multilamellar vescicles was
extruded three times through polycarbonate filters of 400 nm and
five times through 200 nm filters. The final suspension was then
purified by exhaustive dialysis carried out at 4 ꢁC in saline buffer.
The liposomes were characterized by dynamic light scattering
(Zetasizer NanoZS, Malvern, UK) in order to assess the mean hydro-
dynamic size and the polydispersity of the system.
LP1: 72% POPC, 10% cholesterol, 3% DSPE-PEG-methoxy 2000,
15% DPA-C6-(Gd)DOTAMA were dissolved in 10 ml of chloroform/
methanol solution (7/3v/v). The formed thin film was hydratated
with saline buffer and then was extruded. The mean hydrodynamic
diameter was 151nm with a polydispersity index (PDI) of 0.07.
LP2: 87% POPC, 10% cholesterol and 3% DSPE-PEG-methoxy
2000 were dissolved in 10ml of chloroform. The thin film was
hydratated with saline buffer containig DPA-C6-(Gd)DOTAMA
20 mM. The mean hydrodynamic diameter was 149 nm with a PDI
of 0.10
2,20,200-(10-(2-((6-(4-(3-(2-(diethylamino)-2-oxoethyl)-5,7-dimethylpyra-
zolo[1,5-a]pyrimidin-2-yl)phenoxy)hexyl)amino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid (6). To a solution of
5 (0.72 g, 0.72 mmol) in dichloromethane (5 ml), neat trifluoroacetic
acid (0.22 ml, 2.88 mmol) was slowly added and the solution was
stirred at RT for 1 h. The dichloromethane was evaporated and
trifluoroacetic acid (0.22 ml, 2.88 mmol) was added to the residue.
The solution was stirred for 24 h, then diethyl ether was slowly
added (20 ml) to give a yellow-orange solid, which was filtered,
washed with diethyl ether and dried. The crude was purified on
FPLC (Akta Purifier, Pharmacia) on amberchrom CG161 by using iso-
cratic gradient water/methanol as eluent to afford a yellow solid
(0.245 g, 33%). 1H-NMR (CD3OD, 600 MHz) d 7.62 (2H, d, J= 8.4Hz,
Ar), 7.00 (2H, d, J = 8.4Hz, Ar), 6.76 (1H, s, Ar), 4.03 (2H, t, J= 6.6 Hz,
ArOCH2), 3.94 (2H, s, CH2C = O), 3.75–3.30 (8H, b, 3 ꢂ NCH2COOH + 1
ꢂ NCH2CONH), 3.54 (2H, m, NCH2CH3), 3.40 (2H, m, NCH2CH3), 3.21
(2H, t, J = 7.2 Hz, CH2NH-DOTA), 2.74 (3H, s, ArCH3), 2.56 (3H,
s, ArCH3), 3.30–2.00 (16H, b, 8 ꢂ CH2), 1.81 (2H, m, OCH2CH2),
1.60–1.35 (6H, m, 3 ꢂ CH2), 1.24 (3H, t, J= 7.2 Hz, NCH2CH3), 1.12
(3H, t, J= 7.2 Hz, NCH2CH3).13C-NMR (CD3OD, 150 MHz) d 174.7
[2 ꢂ C, CON], 172.3 [C, COOH], 171.8 [C, COOH], 170.0 [C, COOH],
161.1 [C, Ar], 160.0 [C, Ar], 156.5 [C, Ar], 148.8 [C, Ar], 146.9 [C, Ar],
130.9 [2 ꢂ CH, Ar], 126.8 [C, Ar], 115.6 [2 ꢂ CH, Ar], 109.7 [CH, Ar],
101.5 [C, Ar], 69.0 [CH2, OCH2], 57.6 [CH2, NCH2CON], 56.7 [CH2,
NCH2COOtBu], 54.9 [2ꢂ CH2, NCH2COOtBu], 52.3–49.5 [8ꢂ CH2, b,
CH2], 43.6 [CH2, NCH2CH3], 42.0 [CH2, NCH2CH3], 40.5 [CH2], 30.4
[CH2], 30.3 [CH2], 28.9 [CH2], 27.8 [CH2], 26.9 [CH2], 24.2 [CH3, ArCH3],
16.9 [CH3, ArCH3], 14.3 [CH3, NCH2CH3], 13.3 [CH3, NCH2CH3]. MS
ESI + (m/z) 838 [M + H]+, 420 [(M + 2H)/2]+, 280 [(M + 3H)/3]+.
Acknowledgements
This work was supported in part by intramural programs, as well as
the FP6 European Networks of Excellence EMIL (Grant LSHC-CT-
2004-503569) and DiMI (Grant LSHB-CT-2005-512146), and more
recently the European Union’s Seventh Framework Programme
[FP7/2007-2013] INMiND (Grant agreement no. HEALTH-F2-2011-
278850). Part of the present work was also carried out in the frame
of COST TD1004 Action (‘Theranostics’). The authors also wish to
thank Dr Dirk Roeda for proofreading the manuscript and suggest-
ing linguistic corrections.
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Magn. Reson. Chem. 2013, 51, 116–122