Synthesis and testing of a p-H2 hyperpolarized 13C probe based on the pyrazolo[1,5-a]pyrimidineacetamide DPA-713, an MRI vector to target the peripheral benzodiazepine receptors

Article abstract of DOI:10.1002/mrc.2839

DPA-713 is the lead compound of a recently developed 2-phenylpyrazolo[1,5- a]pyrimidineacetamide series that has been shown to display a good targeting capability toward peripheral benzodiazepine receptors, recently renamed translocator protein (18 kDa) o

Full text of DOI:10.1002/mrc.2839

Research Article
Received: 22 July 2011
Revised: 12 September 2011
Accepted: 19 September 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.2839
Synthesis and testing of a p-H2 hyperpolarized
13C probe based on the pyrazolo[1,5-a]
pyrimidineacetamide DPA-713, an MRI vector
to target the peripheral benzodiazepine
receptors
Erika Cerutti,a,b Alessandra Viale,a Annelaure Damont,b Frédéric Dolléb and
Silvio Aimea*
DPA-713 is the lead compound of a recently developed 2-phenylpyrazolo[1,5-a]pyrimidineacetamide series that has been
shown to display a good targeting capability toward peripheral benzodiazepine receptors, recently renamed translocator
protein (18 kDa) or in short TSPO. On the basis of this structure, a novel derivative bearing a [13C]butynoate moiety has been
designed and synthesized (three steps—42% overall yield) providing, upon rapid and quantitative para-hydrogenation, the
corresponding hyperpolarized [13C]alkene. Para-hydrogen-induced polarization effects have been detected in both 1H and
13C-NMR spectra. Upon applying a field cycling procedure, the spin order of para-H2 added hydrogens is transferred on the
13 C carboxylate moiety yielding a signal enhancement of approximately 4500 times. T1 of the carboxylate carbon atom is
approximately 21.9 s (at 9.37 T). A 13 C-MR image has been acquired by using the 13 C RARE (Rapid Acquisition by Relaxation
Enhancement) acquisition protocol on a 10-mM solution. The main limitation to the in vivo use of this novel para-hydrogenated
[
13 C]derivative is its relatively low solubility in aqueous systems. Copyright © 2011 John Wiley & Sons, Ltd.
1
Keywords: NMR; 13C; H; hyperpolarization; para-hydrogen; DPA-713; TSPO
In principle, one may think of introducing a hydrogenatable
Introduction
synthon containing a long T1 carbon center in any given molecule
whose biodistribution/transformation has to be assessed. This
may be compared with the approach used in conventional nuclear
medicine (notably single photon emission tomography), where the
molecule of interest is often modified with an appropriate chelating
agent and a radioactive atom. We have recently proposed such an
approach by conjugating glucose to a hydrogenatable synthon
that contains a long T1 functionality.[25] Here, a second example
of this approach is proposed. It deals with the synthesis and testing
of a novel compound coded DPA-713-[13 C]butynoate, a carbon-
13-enriched butynoic ester derivative of the lead compound
Much attention has been devoted in recent years to the develop-
ment of hyperpolarized carbon-13-containing molecules for
13 C-magnetic resonance imaging.[1–25] In this regard, the most
challenging application deals with their use as metabolic imaging
reporters. In this context, much attention has been devoted to
hyperpolarized [13 C]pyruvate, which has been proposed as a probe
to assess the increased metabolic activity of tumor cells.[10,12,13,15,19]
Beside pyruvate, other metabolites are currently under scrutiny for
different metabolic applications.[14,16–18,20,21,24]
Two procedures can be used to prepare 13 C-hyperpolarized
molecules, namely, Dynamic Nuclear Polarization (DNP) [9,10,12–21,24]
and Para-Hydrogen-Induced Polarization (PHIP),[1–5,11,14,25,26] the
latter being cheaper and of easier applicability because it does
not involve the very low temperatures needed in the Dynamic
Nuclear Polarization method. In fact, it consists of the catalytic
para-hydrogenation of an unsaturated molecule, followed by
transformation of para-hydrogen spin order into net heteronuclear
hyperpolarization. Clearly, the limited number of available
substrates constitutes a drawback of the technique. Unsaturated
substrates, which are readily hydrogenated to yield water-soluble
products, are in fact necessary. Furthermore, they must contain a
13 C nucleus, characterized by long relaxation time (carbonyl or
quaternary carbon atom), which will be scalarly coupled with the
added para-hydrogen atoms in the product molecule.
(DPA-713) of
a recently developed 2-phenylpyrazolo[1,5-a]
pyrimidineacetamide series that has been shown to display a good
targeting capability toward peripheral benzodiazepine receptors,
recently renamed translocator protein (18 kDa) or in short TSPO.[27]
*
Correspondence to: Aime, Silvio, Dipartimento di Chimica IFM and Centro di
Imaging Molecolare, Università degli Studi di Torino, V. Nizza 52, 10126 Torino,
Italy. E-mail: silvio.aime@unito.it
a Dipartimento di Chimica IFM and Centro di Imaging Molecolare, Università
degli Studi di Torino, V. Nizza 52, 10126 Torino, Italy
b CEA, I2BM, Service Hospitalier Frédéric Joliot, 4 Place du Général Leclerc,
F-91406 Orsay, France
Magn. Reson. Chem. 2011, 49, 795–800
Copyright © 2011 John Wiley & Sons, Ltd.

E. Cerutti et al.
The butynoic moiety is readily hydrogenated with para-H2, and
the resulting para-hydrogen-induced polarization is transferred to
the 13C resonance of the ester functionality. Noteworthy, DPA-713
has recently been labeled also with the short-lived positron-emitter
carbon-11 (T1/2 : 20.38 min) as well as functionalized and radiola-
beled at the same structural position (fluoroethoxy group) with
another positron-emitter (fluorine-18, T1/2 : 109.8min) for Positron
Emission Tomography (PET) imaging.[27–34]
the propylalcohol 4 in 68% yield. Coupling with 2-butynoic acid
(CH3-CΞC-COOH) or its 13 C-enriched version (CH3-CΞC-13COOH)
afforded DPA-713-butynoate (1a) and DPA-713-[13C]butynoate (1b)
in 80% and 66% yield, respectively.
Hydrogenation and para-hydrogenation of DPA-713-
butynoate (1a) and DPA-713-[13C]butynoate (1b)
Hydrogenation of 1a (10–50 mM) was performed in acetone-d6
solution, with the addition of a few microliters of CDCl3 to favor
substrate dissolution, directly in the NMR tube (Scheme 2).
[Rh(cyclooctadiene)(diphenylphosphinobutane)][BF4] was used
as catalyst, and the reaction was carried out using a pressure
of 8 atm of H2 at 80 ꢀC, yielding the corresponding alkene
molecule 5a in 100% yield after 10 s (Scheme 2).
Para-hydrogenation was carried out under the same experi-
mental conditions (using both 1a and 1b) and in ALTADENA
modality, affording strongly enhanced signals, both in the 1H and
in the 13 C spectra of the para-hydrogenated product. The 1H
spectrum of 5a, recorded immediately after para-hydrogenation
of 1a, is shown in Fig. 1, whereas the 13C spectrum of 5b, recorded
immediately after para-hydrogenation of 1b, is reported in Fig. 2.
The 1H pattern is consistent with what was previously found in
the para-hydrogenation of other butynoic acid esters:[25,26] three
Results and Discussion
Synthesis of DPA-713-butynoate (1a) and DPA-713-[13 C]
butynoate (1b).
The synthesis of DPA-713-butynoate (1a) and DPA-713-[13C]
butynoate (1b) is reported in Scheme 1. Both molecules were
prepared starting from the pyrazolo[1,5-a]pyrimidineacetamide
DPA-713 (2) (synthesized according to literature procedure[27]
)
in three chemical steps. Demethylation of DPA-713 (2) was
carried out in a 1-M BBr3 solution in dichloromethane (5Eq) at
low temperature (from À60ꢀC to À20 ꢀC), giving the phenol 3 in
a high yield (95%). Subsequently, reaction with 3-bromopropan-
1-ol (1.2 Eq), in DMSO, in the presence of NaOH (2 Eq) as a base, gave
Scheme 1. Synthesis of DPA-713-butynoate (1a) and DPA-713-[13 C]butynoate (1b) from DPA-713 (2). (i) BBr3, CH2Cl2, -60 ꢀC to À20 ꢀC, 2 h at À20 ꢀC; (ii)
NaOH, 3-bromopropan-1-ol, DMSO, 50 ꢀC, 24 h; (iii) CH3CCCOOH, DCC, DMAP, CH2Cl2, RT, 3 h; (iv) CH3CC13COOH, DCC, DMAP, CH2Cl2, RT, 3 h.
Scheme 2. Hydrogenation of DPA-713-butynoate (1a) and DPA-713-[13 C]butynoate (1b). (i) H2 (or para-H2) (8 Atm), [Rh(cyclooctadiene)(diphenylpho-
sphinobutane)][BF4], CDCl3, acetone-d6, 80 ꢀC, 10 s.
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Magn. Reson. Chem. 2011, 49, 795–800

p-H2 hyperpolarized 13C-labeled DPA-713
observed for the 13C carbonyl resonance of the alkene molecule
at 166.18 ppm (Fig. 2) due to polarization transfer from the added
protons to the 13C nucleus via scalar couplings.
The high-intensity enhancement has been exploited for the
acquisition of 13C images of tubes containing hyperpolarized
5b. Images were acquired by using a single shot RARE sequence
on tubes containing different concentrations of the compound,
immediately after para-hydrogenation and field cycling applica-
tion, to determine the minimum detectable concentration, which
turned out to be approximately 10 mM (Fig. 3).
The result is quite promising but one must take into account
two important factors for the potential employment of 5b as
hyperpolarized contrast agent for MRI: (i) the 13C relaxation time
of the carbonyl carbon atom must be long enough to allow the
maintenance of a high degree of polarization even after further
manipulation of the sample, injection, and eventual binding to
the target protein; and (ii) to get a concentration value of
10 mM after injection, a tenfold more concentrated solution must
be injected.
Figure 1. 1H spectrum of derivative 5a recorded immediately after
para-hydrogenation of DPA-713-butynoate (1a (acetone-d6, RT, 9.37 T)).
The 13C relaxation time of the carbonyl carbon atom was
measured at 9.37 T and was found to be 21.9 s. It seems that this
value is not so long when compared with relaxation times of
other acid derivatives, which are currently used as hyperpolarized
agents such as pyruvate, for which the values range around 60 s.
This is mainly due to the molecular size of 5b. Nevertheless, the
measured relaxation time is quite similar to that previously
reported for glucose derivatives of butynoic acid[25] and should,
as in that case, allow to carry out some manipulations for the
preparation of the sample after para-hydrogenation. Further-
more, as the determinant responsible for the relaxation of the
13C ester functionality is its chemical shift anisotropy (approxi-
mately 120 ppm), the T1 value should further increase at lower
fields, which are most often encountered in MRI scanners.
Nevertheless, 10 mM is the minimum concentration detectable
in an MR image recorded immediately after para-hydrogenation,
without further manipulations. Even assuming that all the polari-
zation may be preserved, at least 100 mM of hyperpolarized
5b should be prepared for injection. This means that either
a 100-mM acetone solution should be injected as it is (but
this seems unacceptable for toxicological reasons) or a more
concentrated solution should be hydrogenated and then
diluted in an aqueous system to make it biologically tolerable.
Unfortunately, the low solubility of 5b in polar solvents
prevents both the hydrogenation of very concentrated solu-
tions and the successive dilution in water/saline for injection.
Figure 2. 13C spectra of derivative 5b recorded (a) immediately after
para-hydrogenation of DPA-713-[13 C]butynoate (1b) and field cycling
application and (b) after relaxation (acetone-d6, RT, 14 T).
enhanced peaks corresponding to the methyl protons (emission,
2.22 ppm), Ha (absorption, 6.53 ppm), and Hb (low-intensity
emission, 5.94 ppm), respectively, are observed, the last being
less enhanced than the other two. The pattern can be easily
explained on the basis of the distribution of the spins among
the different spin levels, as reported by Reineri et al.[25] As the hy-
drogenation reaction takes place outside the spectrometer
(ALTADENA experiment), all protons resonate at the same Larmor
frequency, and the strong coupling condition among them is
achieved. As a consequence, the spin states of all the protons
are mixed. The population of each level is a function of the
para-hydrogen contribution to that state and can be evaluated
by a numerical approach: it results that the overpopulation
derived from para-hydrogen is concentrated in the lowest energy
spin states having Mz = ½ and Mz = À½, respectively, and the
adiabatic transfer of the sample into the spectrometer maintains
the same spin states population pattern. The resulting enhanced
transitions involve the methyl proton (emission) and Ha (adsorp-
tion), whereas the Hb transition occurs between levels that are
almost equally populated, thus resulting in low signal enhancement.
The 13C spectrum was recorded immediately after para-
hydrogenation and application of a field cycling procedure. The
latter consists of quickly taking the sample to a quasi-zero
magnetic field and then slowly bringing it back to earth magnetic
field.[2,26] A strong signal enhancement (approximately 4500
times with respect to the equilibrium signal intensity) was
Figure 3. Single-shot 13 C RARE images of tubes containing (a) 10 mM
and (b) 7 mM hyperpolarized 5b in acetone-d6 (7 T, axial section).
Smoothing has been applied to the images for publication: the reported
images are 364 Â 364.
Magn. Reson. Chem. 2011, 49, 795–800
Copyright © 2011 John Wiley & Sons, Ltd.
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E. Cerutti et al.
and 150 MHz for 13C and on a JEOL EX-400 spectrometer operat-
Conclusions
1
ing at 400 MHz for H. T1 measurements were carried out on a
degassed sample of the 13C-enriched compound by using the
inversion recovery pulse sequence.
The 2-phenylpyrazolo[1,5-a]pyrimidineacetamide DPA-713 was
successfully derivatized and coupled to both a 13 C-enriched
and a non-isotopically enriched butynoate moiety. These novel
derivatives were compatible with rapid and quantitative hydro-
genation methods and did efficiently incorporate the two hydro-
gen atoms of para-H2, giving access to a 13 C hyperpolarized
probe by a field cycling procedure. A signal enhancement of
approximately 4500 times for the 13C carbonyl resonance was
observed. T1 of the 13C carbonyl moiety in the DPA-713-butenoate
derivative reported herein seems significantly decreased with
respect to the parent methyl butenoate molecule. The major
drawback for further in vivo applications is, however, the very
poor solubility of this derivative in water. The short lifetime of
the probe, together with the need for reaching concentrations
at the targeting region in the (high) millimolar range, may not
facilitate the use of the herein reported probe for in vivo
applications. Work is currently in progress to circumvent some
of the limitations described earlier.
Single scan 13C images were obtained on a Bruker Avance 300
spectrometer (7 T) equipped with a Microimaging probe, using
the RARE sequence, with a RARE factor of 32, matrix 32 Â 32, echo
time 8.465 ms.
Chemistry
N,N-diethyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)
acetamide (DPA-713, 2)
Synthesized according to James et al.[27]
1
Rf (CH2Cl2/CH3OH, 95:5 v/v): 0.39 H-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 Â CH, Ar], 127.0 [C, Ar], 114.4 [2 Â 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) 367.1 [M + H]+.
Experimental
Chemicals were purchased from Sigma-Aldrich and used with no
further purification. 13 C-enriched 2-butynoic (99% enrichment)
acid was prepared according to the published procedure.[25]
Solvents were stored over molecular sieves and purged with
nitrogen before use. Hydrogen was produced by a CLAIND
generator, model HG300. Para-enriched hydrogen (52 %) was
prepared storing H2 over Fe2O3 at 77ꢀK for 1 h.
N,N-diethyl-2-(2-(4-hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-
3-yl)acetamide (3)
A solution of BBr3 (1 M in CH2Cl2, 60 ml) was added dropwise at
À60 ꢀC under argon atmosphere to a solution of compound 2
(4.39 g, 11.98 mmol) in 50 ml of CH2Cl2. The temperature was
slowly increased to À20 ꢀC, and the reaction mixture was stirred
at this temperature for 2 h. The reaction was quenched with
methanol (25 ml) and neutralized with aqueous NH3 solution.
The formed precipitate was collected and washed with water
(2 Â 10 ml). A pale yellow solid (4.04 g, 95%) was obtained.
Chromatographies were conducted on silica gel (0.63-0.200 mm,
VWR) columns. Thin layer chromatography was carried out using
aluminum precoated plates of silica gel 60F254 (VWR). Compounds
were detected using a UV lamp (254 nm) and by sprinkling the thin
layer chromatography plate with KMnO4 aqueous solution and
heating.
1H-NMR and 13C-NMR spectra for characterization of synthesis
products were recorded on a Bruker (Wissembourg, France) Avance
(400 MHz) apparatus using the hydrogenated residue of the deuter-
ated solvents CDCl3 (d = 7.26 ppm) or CD2Cl2 (d = 5.32 ppm) for
1H-NMR as well as the deuterated solvents CDCl3 (d = 77.2 ppm)
or CD2Cl2 (d = 54.0ppm) 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 electrospray ionization (ESI+)).
Para-hydrogenation reactions were carried out in ALTADENA
conditions in a 5-mm NMR tube equipped with a Young valve.
[Bis(diphenylphosphino)butane](1,5-cyclooctadiene)rhodium(I)
tetrafluoroborate (5–15 mg) was dissolved in acetone-d6 (0.3 ml)
and activated by reaction with H2. Normal H2 was then replaced
by the para-H2 enriched mixture (4 atm), after addition of the
substrate (0.1 ml of 5–50 mM acetone-d6 solutions containing
10 ml of CDCl3 to favor substrate dissolution). The hydrogenation
was carried out by shaking the tube for 10 s.
1
Rf (CH2Cl2/CH3OH, 92:8 v/v): 0.41. H-NMR (CD2Cl2, 400 MHz) d
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 Â CH, Ar], 125.4 [C, Ar], 116.0 [2 Â 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) 353.1 [M + H]+.
N,N-diethyl-2-(2-(4-(3-hydroxypropoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]
pyrimidin-3-yl)acetamide (4).
DMSO (30 ml), NaOH (3.40 g, 2.04 mmol), and 3-bromopropan-
1-ol (0.18 ml, 2.04 mmol) were added to a solution of compound
3 (0.60 g, 1.70 mmol). The solution was stirred at 50 ꢀC overnight.
After cooling to room temperature, the reaction was quenched
with a saturated aqueous solution of K2CO3, and extracted with
ethyl acetate (2 Â 50 ml). The combined organic layers were
washed with brine (4 Â 50 ml), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude was purified
on silica gel (heptane–acetone, 2:1 v/v) to afford product 4
(0.47 g, 68%) as a white solid.
The magnetic field cycle was pursued by quickly inserting the
tube into a m-metal shield (field strength 0.1 mT) and then slowly
removing the shield (the entire field cycling procedure required
3 to 5 s).
For para-H2 experiments, single scan spectra were acquired on
1
a Bruker Avance600 spectrometer, operating at 600 MHz for H
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Copyright © 2011 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2011, 49, 795–800

p-H2 hyperpolarized 13C-labeled DPA-713
Rf (heptane–acetone, 1:2 v/v): 0.35. 1H-NMR (CD2Cl2, 400 MHz) d
7.72 (2H, d, J = 8.8 Hz, Ar), 6.99 (2H, d, J = 8.8 Hz, Ar), 6.54 (1H, s,
Ar), 4.15 (2H, t, J = 6.0 Hz, ArOCH2), 3.88 (2H, s, CH2CO), 3.81 (2H,
m, CH2OH), 3.50 (2H, q, J = 7.2 Hz, NCH2CH3), 3.38 (2H, q,
J = 7.2 Hz, NCH2CH3), 2.72 (3H, s, ArCH3), 2.53 (3H, s, ArCH3), 2.03
(2H, m, HOCH2CH2), 1.84 (1H, m, OH), 1.22 (3H, t, J = 7.2 Hz,
NCH2CH3), 1.11 (3H, t, J = 7.2Hz, NCH2CH3). 13C-NMR (CD2Cl2,
100 MHz) d 170.3 [C, CO], 159.7 [C, Ar], 158.2 [C, Ar], 155.0 [C, Ar],
148.3 [C, Ar], 145.4 [C, Ar], 130.2 [2 Â CH, Ar], 127.0 [C, Ar], 114.9
[2 Â CH, Ar], 108.7 [CH, Ar], 101.2 [C, Ar], 66.2 [CH2, ArOCH2], 60.5
[CH2, CH2OH], 42.8 [CH2, NCH2CH3], 41.0 [CH2, NCH2CH3], 32.7
[CH2, OCH2CH2CH2OH], 28.6 [CH2, CH2CO], 24.9 [CH3, ArCH3], 17.2
[CH3, ArCH3], 14.7 [CH3, NCH2CH3], 13.4 [CH3, NCH2CH3]. MS ESI +
(m/z) 411.2 [M + H]+, 433.2 [M+ Na]+, 843.0 [2M + Na]+.
[2 Â CH, Ar], 126.7 [C, Ar], 114.7 [2 Â CH, Ar], 108.3 [CH, Ar],
100.9 [C, Ar], 85.9 [C, d, J = 21 Hz, CΞCCH3], 72.4 [C, d, J = 127 Hz,
CCΞCH3], 64.2 [CH2, ArOCH2], 62.7 [CH2, OCH2CH2CH2], 42.4
[CH2, NCH2CH3], 40.7 [CH2, NCH2CH3], 28.5 [CH2, d, J = 2 Hz, CH2O-
COR], 28.3 [CH2, CH2CO], 24.8 [CH3, ArCH3], 17.0 [CH3, ArCH3], 14.5
[CH3, NCH2CH3], 13.2 [CH3, NCH2CH3], 3.9 [CH3, d, J = 2 Hz,
CCΞCH3]. MS: ESI + (m/z) 478.21 [M + H]+, 500.2 [M + Na]+.
Acknowledgements
This work was supported in part by the EC-FP6-projects DiMI (LSHB-
CT-2005-512146) and EMIL (LSH-2004-503569). The authors grate-
fully acknowledge Regione Piemonte (bando POR-FESR 2007, Asse
1, Misura I.1.1) for financial support. The authors also thank Dr Dirk
Roeda for proof reading the manuscript and suggesting linguistic
corrections and Dr Walter Dastrù for the acquisition of MR images.
3-(4-(3-(2-(Diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-
2-yl)phenoxy)propyl but-2-ynoate (1a).
To a solution of compound 4 (0.46 g, 1.12 mmol) and 2-butynoic References
acid (0.10 g, 1.23 mmol) in 10 ml of CH2Cl2 DCC (0.25 g,
[1] K. Golman, O. Axelsson, H. Johannesson, S. Mansson, C. Olofsson,
1.23 mmol) dissolved in 5 ml of CH2Cl2 was added dropwise at
0 ꢀC under argon atmosphere. The solution was stirred for 5 min
at 0 ꢀC, and then a catalytic amount of DMAP was added. The
reaction was stirred at room temperature for 3 h. The precipitated
dicyclohexyl urea was filtered off, and the solvent was evaporated
under reduced pressure. The crude was purified on silica gel
(eluent: heptane–acetone, 80:20 v/v). The obtained solid was trit-
urated in diethyl ether to afford pure product 1a (0.44 g, 80%).
Rf (heptane–acetone, 50:50 v/v): 0.32. 1H-NMR (CDCl3, 400 MHz)
d 7.76 (2H, d, J = 8.8 Hz, Ar), 6.97 (2H, d, J = 8.8 Hz, Ar), 6.50 (1H, s,
Ar), 4.37 (2H, t, J = 6.0 Hz, CH2OCOR), 4.10 (2H, t, J = 6.0 Hz,
ArOCH2), 3.91 (2H, s, CH2CO), 3.50 (2H, q, J = 7.2 Hz, NCH2CH3),
3.41 (2H, q, J = 7.2 Hz, NCH2CH3), 2.73 (3H, s, ArCH3), 2.53 (3H, s,
ArCH3), 2.17 (2H, m, OCH2CH2), 1.99 (3H, s, CCΞCH3), 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, CO], 159.0 [C, Ar], 157.6 [C, Ar],
155.1 [C, Ar], 153.8 [C, OCOR], 147.8 [C, Ar], 144.8 [C, Ar], 130.1
[2 Â CH, Ar], 126.7 [C, Ar], 114.7 [2 Â CH, Ar], 108.3 [CH, Ar],
100.9 [C, Ar], 85.9 [C, CΞCCH3], 72.5 [C, CΞCCH3], 64.2 [CH2,
ArOCH2], 62.7 [CH2, OCH2CH2CH2], 42.4 [CH2, NCH2CH3], 40.7
[CH2 NCH2CH3], 28.5 [CH2, CH2OCOR], 28.3 [CH2, CH2CO], 24.8
[CH3, ArCH3], 17.0 [CH3, ArCH3], 14.5 [CH3, NCH2CH3], 13.2 [CH3,
NCH2CH3], 4.0 [CH3, CΞCCH3]. MS ESI + (m/z) 477.4 [M + H]+,
499.3 [M + Na]+.
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Magn. Reson. Chem. 2011, 49, 795–800