Chemical delivery system of metaiodobenzylguanidine (MIBG) to the central nervous system

Article abstract of DOI:10.1021/jm901550z

The aim of the present investigation was to apply a chemical delivery system (CDS) to MIBG(4) with the purpose of delivering this drug to the CNS. Compound 4 has been linked to a 1,4-dihydroquinoline moiety in order to achieve its CNS penetration, and here we report the synthesis to link 4 to the chemical delivery system and the radiosynthesis with carbon-11 of the CDS-4 entity . After iv injection into rats of the [¹¹C]CDS-4, the follow-up study of the radioactivity distribution in blood samples and brain homogenates and the analysis by HPLC and LC-MS/MS have confirmed the release of 4 into the CNS.

Full text of DOI:10.1021/jm901550z

J. Med. Chem. 2010, 53, 1281–1287 1281
DOI: 10.1021/jm901550z
Chemical Delivery System of Metaiodobenzylguanidine (MIBG) to the Central Nervous System
†
†
†
†
‡
Fabienne Gourand, Guillaume Mercey, Meziane Ibazizene, Olivier Tirel, Joel Henry, Vincent Levacher, Cecile Perrio,
ꢀ
and Louisa Barre*
§
†
ꢀ
ꢁ
€
ꢀ
,†
†
Groupe de Deꢀveloppements Meꢀthodologiques en Tomographie par Emission de Positons, CEA/DSV/I2BM/CI-NAPS UMR6232, Universiteꢀde
Caen Basse Normandie, Caen, France, ‡UMR 100 IFREMER, Physiologie et Ecophysiologie des Mollusques Marins, Universiteꢀde Caen, France,
and Laboratoire de Chimie Organique Fine et Heꢀteꢀrocyclique, UMR 6014, IRCOF, CNRS, Universiteꢀ et INSA de Rouen. B.P. 08 F-76131
Mont-Saint-Aignan Cedex, France
§
Received October 19, 2009
The aim of the present investigation was to apply a chemical delivery system (CDS) to MIBG (4) with the
purpose of delivering this drug to the CNS. Compound 4 has been linked to a 1,4-dihydroquinoline
moiety in order to achieve its CNS penetration, and here we report the synthesis to link 4 to the chemical
delivery system and the radiosynthesis with carbon-11 of the “CDS-4 entity”. After iv injection into rats
of the [11C]CDS-4, the follow-up study of the radioactivity distribution in blood samples and brain
homogenates and the analysis by HPLC and LC-MS/MS have confirmed the release of 4 into the CNS.
Introduction
for brain imaging. To the best of our knowledge, only one
paper has already investigated the potential of this CDS
approach to improve the delivery of radiolabeled agents into
the CNS.9 However, the relative instability of the dihydropyr-
idine carrier used in this investigation appeared also to be a
major limitation, hampering severely the development of this
CDS strategy. In this context, the aim of the present investiga-
tion was to apply the dihydroquinoline/quinolinium salt type
CDS to the m-iodobenzylguanidine (MIBG) with the purpose
of delivering this drug to the CNS. [123I/131I]MIBG imaging is
the most accurate method for detection of catecholamine-
secreting tumors including neuroendocrine tumors such as
pheochromocytoma, paraganglioma, neuroblastoma, carci-
noid, and many medullary thyroid cancers.10 In addition to its
application as a marker of adrenergic tumors, [123I]MIBG has
proven to be very useful for scintigraphic imaging studies
of cardiac sympathetic innervation.11 MIBG is a guanethidine
derivative structurally resembling norepinephrine. Studies
have demonstrated that MIBG is actively taken up into
human neuroblastoma cells by the norepinephrine transpor-
ter (NET) and that MIBG uptake was attenuated into
neuroblastoma cells in the presence of imipramine, an inhi-
bitor of NET.12,13 Cellular accumulation of MIBG occurs by
two distinct mechanisms: an active uptake, which is specific,
high-affinity, saturable, and ATPase-dependent and only
occurs in cells that synthesize the NET, and a passive diffu-
sion, which is nonspecific, low-level, energy-independent, and
unsaturable and takes place in all cells.14 In addition, MIBG is
concentrated into storage vesicles via vesicular monoamine
transporter (VMAT) and MIBG uptake can be inhibited with
pretreatment with drugs such as reserpine, a specific inhibitor
of VMAT.15
Drug delivery to the brain is often limited by the BBB,
which regulates the exchange of substances between the peri-
pheral circulation and the central nervous system (CNSa).
Various attempts have been made to overcome the limited
access of drugs into the brain. One of these strategies was the
linking of an active drug to a brain-specific carrier, which
delivers the drug specifically into the brain, where it is cleaved
enzymatically from the carrier. The 1,4-dihydropyridine/pyri-
dinium salt redox-chemical delivery systems have been used
by Bodor to improve the access of therapeutic agents to the
central nervous system.1-4 The principle of the CDS is illu-
strated in Scheme 1. These chemical delivery systems (CDSs)
based on the redox conversion of a lipophilic dihydropyridine
to an ionic lipid-insoluble pyridinium salt are equivalent to the
NADH/NADþ oxidation system. The important criteria for a
CDS is that it should be sufficiently lipophilic to enter the
central compartment and should then undergo an enzymatic
conversion to promote drug retention in the CNS. Usually,
the drug was conjugated through ester or amide bond to a
carboxylic function of the carrier system. Nevertheless, the
main drawback observed with the dihydropyridines is their
instability against oxidation and mainly hydration of the
5,6-double bond.5,6 The modest stability of this class of car-
riers remains an obstacle to further development of this CDS
approach, and a possibility to overcome this problem was to
replace the dihydropyridine/pyridinium salt system by a
dihydroquinoline/quinolinium salt system.7,8
The delivery of radiopharmaceuticals to the CNS would
constitute another interesting application of this redox CDS
*To whom correspondence should be addressed. Phone:
þ33231470224. Fax: þ33231470275. E-mail: barre@cyceron.fr.
a Abbreviations: CDS, chemical delivery system; CNS, central ner-
vous system; MIBG, metaiodobenzylguanidine; BBB, blood-brain
barrier; NET, norepinephrine transporter; VMAT, vesicular mono-
amine transporter; ADHD, attention deficit hyperactivity disorders;
EOS, end of synthesis; PET, positron emission tomography; SPECT,
single photon emission computed tomography.
The NET is involved in several neuropsychiatric disorders
and is a molecular target for the treatment of depression,
attention deficit hyperactivity disorders (ADHD), and anxi-
ety disorders.16 MIBG does not cross the BBB, and it has been
demonstrated in vitro that [125I]MIBG maps the monoamine
storage and release sites on rat brain slices.17 A CDS which
r
2010 American Chemical Society
Published on Web 01/19/2010
pubs.acs.org/jmc

1282 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Scheme 1. Bodor’s Chemical Delivery System (CDS)
Gourand et al.
dithionite and sodium carbonate gave 3 in only a 20-52%
moderate yield.28 In contrast, the use of BNAH (1-benzyl-1,4-
dihydronicotinamide) provided the corresponding final target
3 with an excellent yield of 95%.29
Radiochemistry. The radiosynthesis of [11C]3 was carried
out as illustrated by Scheme 3. The quaternarization reaction
of the quinoline 1 with [11C]methyl triflate afforded the
quaternary salt [11C]2 with an incorporation of [11C]-
CH3OTf up to 95%. Reduction of [11C]2 was made by
treatment with BNAH and provided the corresponding
1,4-dihydroquinoline [11C]3 in an excellent radiochemical
yield of 95%. After HPLC purification and formulation
[11C]3 was obtained with a radiochemical purity greater than
99% and specific activities ranging from 13 to 24 GBq/μmol
as evaluated by analytical HPLC at the end of synthesis
(EOS).
In Vitro Stability of [11C]3 in Rat Blood Samples. The
chemical stability of [11C]3 toward oxidation was first in-
vestigated in vitro on blood samples to establish whether it is
stable enough to start developing an ex vivo evaluation. To
carry out this investigation, [11C]3 was added to rat blood,
and after an efficient preparation process where >90% of
the radioactivity was recovered, the plasma samples were
analyzed using reverse phase HPLC analysis. The chroma-
tograms obtained at various time points (5, 15, 30, and 60
min) did not show any trace of the quaternary form-[11C]2.
Additionally, no byproduct which could have arisen from
hydration reactions were detected, indicating that [11C]3
exhibit a good stability in rat blood.
In Vivo Evaluation. The aim of this work was to study the
feasibility of using the 1,4-dihydroquinoline/quinolinium
salt redox system as a potential chemical delivery system of
4 to the CNS. As shown in Scheme 4, we have evaluated
the penetration of [11C]3 through the blood-brain barrier
(BBB), its brain oxidation to the corresponding quaternary
form-[11C]2 expected in vivo, and finally the release of 4 and
[11C]5 after enzymatic cleavage of the quinolinium salt. The
radioactivity kinetics of rat brain uptake were determined in
vivo after [11C]3 injection following the sacrifice of animals at
different time intervals over a period of 60 min. The radio-
activity brain uptake was rapid, peaking at 0.10% ID/g at
3 min and declining to 0.06% ID/g at 60 min (Figure 1).
These data indicate a moderate penetration of [11C]3 through
the BBB with a progressive and slow elimination of the brain
radioactivity.
According to the CDS concept described earlier, the
oxidation process is very crucial to predict the ability of
the 1,4-dihydroquinoline CDS to release the compound in
the brain. A fast oxidation process will convert the CDS
into the corresponding quaternary salt in the blood before
reaching the brain. A satisfactory compromise should be
obtained between the stability of the 1,4-dihydroquinoline
species to ensure its survival in the blood and its subsequent
oxidation in the brain. To clearly identify the radioactive
species in the brain and blood, respectively, and to provide in
vivo information about the [11C]3 oxidation kinetics into its
corresponding quaternary form-[11C]2, samples were treated
so as to recover >90% of the radioactivity and then ana-
lyzed by radioHPLC over a period of 60 min after [11C]3
injection in rat.
would permit the crossing of MIBG through the BBB would
provide a potential imaging marker of monoamine uptake,
storage, and release in the brain with a wide field of applica-
tion in neurodegenerative or neuropsychiatric disorders.
Previously, we have labeled with carbon-11 (t1/2 = 20.4
min) a dihydroquinoline derivative, chosen as a model for
chemical carrier in order to validate the concept of CDS
described above.18 In vivo evaluation of the N-[11C-methyl]-
dihydroquinoline was investigated in rats after iv injection
where a rapid penetration into the CNS followed by a fast
oxidation into the [11C]quinolinium salt was identified. These
encouraging results have demonstrated that this CDS ap-
proach could be used as a potential delivery system for the
transport of drug which does not cross the BBB. For this
reason, 4 has been linked to a 1,4-dihydroquinoline moiety in
order to achieve its CNS penetration, and here we report the
synthesis to link 4 to the chemical delivery system and the
radiosynthesis with carbon-11 of the “CDS-4 entity”. After iv
injection into rats of the [11C]CDS-4, the distribution of the
radioactivity was followed in blood samples and brain homo-
genates and analyzed by HPLC and LC-MS/MS.
Results and Discussion
Chemistry. The synthesis of N-(3-iodobenzyl)-N0-(1-methyl-
3-carbonyl-1,4-dihydroquinoline)guanidine (3) was accom-
plished as described in Scheme 2. The key intermediate N-(3-
iodobenzyl)-N0-(3-carbonyl-quinoline)-guanidine (1) was
prepared using four different routes by reaction of several
activated esters of 3-quinoline carboxylic acid with 4. The
first attempts involving the condensation of 4 with the
corresponding methyl ester of 3-quinoline carboxylic acid
or with a crotonamide intermediate obtained with N-tert-
butyl-5-methylisoxazolium perchlorate (NBI) provided 1 in
35-37% yield.19-25 To find a better procedure, we have also
used the N,N0-carbonyldiimidazole (CDI) or N-hydroxysuc-
cinimide as carboxylate activating agents.26 The reactions of
resulting activated esters with 4 showed a significant increase
in reactivity and compound 1 was isolated in 59.5-61.5%
yield. An alternative route to obtain 1 via the acid chloride of
3-quinoline carboxylic acid and the guanidine was unsuc-
cessful.27 The quaternarization reaction of 1 with methyl
trifluoromethanesulfonate afforded N-(3-iodobenzyl)-N0-(1-
methyl-3-carbonyl-quinolinium)-guanidine trifluoromethane
sulfonate (2) in 50% yield. In the final step, the regioselective
reduction of 2 performed with a large excess of sodium
HPLC analysis of brain extracts showed two radioactive
peaks, one corresponding to [11C]3 (tR = 13 min) and the
other to a polar radioactive compound (tR = 3.5 min). The
percentage of unchanged [11C]3 declined from 96% at 3 min

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1283
Scheme 2. Synthesis of 3a
a Reagents and conditions: (a) H2SO4, MeOH, 100 °C, 12 h then 4/DMF/100 °C, 3 h; (b) NBI, NEt3, DMF, rt, 4 h then 4, DMF, 140 °C, 2 h; (c) CDI,
DMF, rt, 1 h then 4, DMF, rt, 12 h; (d) HOSu, THF, DCC, rt, 12 h then 4, DMF, 100 °C, 2 h; (e) MeOTf, CH2Cl2, rt, 30 min; (f) BNAH, CH2Cl2, rt, 12 h.
Scheme 3. Radiosynthesis of [11C]3a
a Reagents and conditions: (a) [11C]MeOTf, CH3CN, rt, 7 min; (b) BNAH, CH3CN, rt, 5 min.
Scheme 4. Chemical Delivery System of 4 into the CNS
to 59% at 60 min while the main polar radioactive compound
appeared, which increased from 4% at 3 min to 41% at
60 min (Figure 2); in parallel, [11C]3 decreased rapidly from
the blood (30% at 3 min) and was not detected beyond 20
min post injection; at this time, only the main polar radio-
active compound (tR = 3.5 min) was found (Figure 3). An
extra and minor radioactive peak corresponding to the
quaternary form-[11C]2 (tR = 7.5 min) has been observed
in the plasma sample only at 3 and 5 min after injection. The
HPLC profiles from brain and plasma extracts let us suggest
an oxidation of [11C]3 into the CNS followed by a rapid
enzymatic cleavage, which could lead to the carboxylic acid
[11C]5 and compound 4 (Scheme 4).
polar radioactive compound. Because of this observation, we
could assign, with confidence, the main polar radioactive
compound as [11C]5. On the basis of these findings, a
complementary study has been conducted to identify by
LC-MS/MS the release of 4 resulting from the amide bond
cleavage of the quaternary form 2. Previously, 4 and com-
pounds 2 and 3 were injected as reference and the data
obtained under our LC-MS/MS conditions gave similar
fragmentation pattern for 2 and 3.30 Then, compound 3
(1 mg) was injected into rat. After sacrifice at 60 min, the
brain was removed and treated, and the sample processing
procedure was sufficient to provide clean filtrates before
injection to the sensitive LC-MS/MS system. A typical
chromatogram from brain extracts is shown in Scheme 5.
The mass spectral analysis revealed molecular ion at m/z 276
assigned to 4 (tR = 10.36 min) and which was confirmed by
MS/MS data, where the fragmentation of the molecular
ion generated specific product ions at m/z 233 and 217; the
To demonstrate this hypothesis, the validity of [11C]5 was
scrutinized by the synthesis of the stable analogue 5. After 5
coinjection onto HPLC with either brain or plasma extracts,
obtained from rat injected with [11C]3, its retention time
(tR = 3.5 min) was revealed to be similar to that of the main

1284 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Gourand et al.
Figure 3. Percentage of unchanged [11C]3, [11C]5, and [11C]2 in rat
plasma (n = 2 for each time point).
Figure 1. Ex vivo biodistribution of the radioactivity in rat brain at
different time intervals following tail vein injection of [11C]3. Results are
expressed in percent of injected dose per gram (%ID/g ( SD, n = 2).
fication. 1H and 13C NMR spectra were measured on a Bruker
DPX 250 at 250 MHz (1H) and 62.5 MHz (13C). Samples were
dissolved in an appropriate deuterated solvent (CDCl3, DMSO-
d6 or CD3OD). Chemicals shifts are reported as parts per million
(δ) relative to tetramethylsilane (TMS, 0.00 ppm), which was
used as an internal standard. Coupling constants are given in
Hz and coupling patterns are abbreviated as: s (singulet),
d (doublet), t (triplet), m (multiplet), dt (doublet of triplet), br
(broad). Elemental analyses were performed on a ThermoQuest
analyzer CHNS and were within (0.44% of the calculated
values. In the radiochemistry procedure, the labeled compounds
were isolated on a HPLC system equipped with Merck L-6200
pump and a Merck L-4250 detector in series with Novelec βþ-
flow detector. For each described labeled compound, the char-
acteristics of the column and solvent used will be described. The
identity of the labeled compounds was confirmed by its coelu-
tion with the unlabeled reference compound onto the HPLC
system. Purity of the tested compounds was determined using
analytical reverse phase HPLC and was found to be more
than 98%.
N-(3-Iodobenzyl)-N0-(3-carbonyl-quinoline)-guanidine (1). (a)
Via CDI. CDI (162 mg, 1 mmol) was added to a solution of
3-quinoline carboxylic acid (173 mg, 1 mmol) in dimethylform-
amide (3.5 mL). The resulting mixture was stirred at room
temperature for 1 h and then was added to a freshly prepared
guanidine base 4 obtained from guanidine hydrochloride 4,HCl
(311 mg, 1 mmol). The mixture was stirred at room temperature
for 12 h. Afterward, acetonitrile (10 mL) was added and the
precipitate was filtered. The resulting filtrate was dried under
reduced pressure, and the residue was suspended in cold water
(10 mL). After one week at 4 °C, the precipitate was filtered,
washed with water, and dried. Compound 1 was obtained in
Figure 2. Percentage of unchanged [11C]3 and [11C]5 in rat brain
(n = 2 for each time point).
molecular ion at m/z 445 was attributed to the parent
compound (tR = 12.76 min) with specific MS/MS fragmen-
tation at 212 and 428. These specific ions were absent in brain
extract from a control rat. These encouraging results have
demonstrated, in vivo, the “proof of concept” that the 1,4-
dihydroquinoline/quinolinium salt redox-chemical delivery
system can be used as an effective tool to deliver drug into the
brain. However, the moderate penetration of [11C]3 through
the blood-brain barrier could be explained by an early
oxidation in the peripheral system before reaching the brain.
The improvement of this chemical delivery system CDS-4 is
based on a balance between the peripheral oxidation of the
1,4-dihydroquinoline carrier and the brain oxidation leading
to the release of 4. To tune the redox potential of the carrier,
additional substituents on the phenyl ring could be easily
installed and the impact of a linker between the carrier and 4
will be investigated.
In conclusion, this study provides the feasibility to use the
1,4-dihydroquinoline/quinolinium salt redox-chemical de-
livery system as a promising tool for the transport of drug
across the blood-brain barrier. This strategy could have a
real application for PET or SPECT imaging where radio-
pharmaceuticals are excluded from transport from blood to
brain.
1
59.5% yield. H NMR (CD3OD): δ 9.51 (s, 1H), 8.95 (s, 1H),
8.00-8.10 (m, 2H), 7.75-7.90 (m, 2H), 7.60-7.70 (m, 2H), 7.41
(d, 1H, J = 7.6 Hz), 7.14 (t, 1H, J = 7.6 Hz), 4.64 (br s, 1.3H),
4.48 (br s, 0.7H). 13C NMR (DMSO-d6): δ 174.1, 161.4, 151.1,
148.8, 143.2, 137.9, 136.8, 136.2, 135.9, 131.7, 130.9, 129.9,
129.0, 127.5, 127.3, 127.1, 95.2, 43.5. Anal. (C18H15IN4O)
C, H, N. (b) via N-(3-carbonyloxy-quinoline)-pyrolidine-2,5-
dione. Under nitrogen, N-hydroxysuccinimide (127 mg, 1.1
mmol) was added to a solution of 3-quinoline carboxylic acid
(173 mg, 1 mmol) in tetrahydrofuran (5 mL) and the mixture was
cooled at -5 °C. A solution of DCC (240 mg, 1.1 mmol) in
tetrahydrofuran (3 mL) was added, and the resulting mixture
was stirred at room temperature overnight. The suspension was
filtered, and the filtrate was evaporated under reduced pressure.
The residue was washed with saturated NaHCO3 and extracted
with chloroform (4 ꢀ 10 mL). The combined organic phases
were dried and evaporated under reduced pressure. N-(3-Car-
bonyloxy-quinoline)-pyrolidine-2,5-dione was obtained in 93%
1
yield as a white solid. H NMR (CDCl3): δ 9.47 (s, 1H), 9.00
(s, 1H), 8.21 (d, 1H, J = 7.7 Hz), 7.97 (d, 1H, J = 7.0 Hz), 7.91
Experimental Section
General. All reagents were purchased from Acros Organics,
Fluka, or Sigma Aldrich and were used without further puri-
(t, 1H, J = 7.0 Hz), 7.69 (t, 1H, J = 7.2 Hz), 2.96 (s, 4H). 13
C
NMR (CDCl3): δ 169.0, 160.9, 150.5, 140.4, 133.1, 129.7, 129.4,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1285
Scheme 5. Analysis of Brain Homogenate in μLC-ESI-MS/MSa
a (A) Total ionic chromatogram. (B) MS spectrum of the peak eluted at 10.36 min. (C) MS/MS spectrum of the m/z 276. (D) MS spectrum of the peak
eluted at 12.76 min. (E) MS/MS spectrum of the m/z 445.
128.1, 126.4, 118.3, 25.6. Then the compound (150 mg, 0.56
mmol) in dimethylformamide (5 mL) was added to guanidine
base 4 obtained from guanidine hydrochloride 4,HCl (175 mg,
0.56 mmol). The mixture was heated at 100 °C for 2 h. Then
solvent was evaporated under reduced pressure, and water
(10 mL) was added to the residue. The resulting mixture was
kept at 4 °C for one week, and the precipitate was filtered,
washed with water, and dried. Compound 1 was obtained in
66% yield.
N-(3-Iodobenzyl)-N0-(1-methyl-3-carbonyl-quinolinium)-guani-
dine Trifluromethanesulfonate (2). Under nitrogen, compound
1 (100 mg, 0.23 mmol) was dissolved in dichloromethane
(50 mL) at 50 °C. The solution was cooled to room temperature,
and methyl trifluoromethane sulfonate (27 μL, 0.23 mmol) was
added. Then the mixture was stirred at room temperature for
30 min. Diethyl ether (50 mL) was added, and after 1 h, the
formed precipitate was collected, washed with diethyl ether, and
dried. Compound 2 was isolated in 50% yield as a beige solid. 1H
NMR (CD3OD): δ 9.84 (s, 1H), 9.68 (s, 1H), 8.61 (d, 1H, J = 8.0
Hz), 8.57 (d, 1H, J = 8.0 Hz), 8.45 (t, 1H, J = 7.6 Hz), 8.18
(t, 1H, J = 7.6 Hz), 7.84 (s, 1H), 7.72 (d, 1H, J = 8.0 Hz), 7.48
(d, 1H, J = 7.6 Hz), 7.20 (t, 1H, J = 7.6 Hz), 4.79 (s, 2H), 4.67
(s, 3H).
N-(3-Iodobenzyl)-N0-(1-methyl-3-carbonyl-1,4-dihydroquino-
line)guanidine(3). Under nitrogen, N-benzyl 1,4-dihydronicoti-
namide (BNAH) (22 mg, 0.1 mmol) was added to a mixture
of quinolinium salt 2 (56 mg, 0.1 mmol) in dichloromethane
(20 mL). The solution was stirred at room temperature for 12 h.
The organic layer was washed three times with water and
evaporated under reduced pressure. Compound 3 was obtained

1286 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Gourand et al.
and water until experiment time. Anesthesia was induced
with 5% isoflurane in a gas mixture of nitrous oxide/oxygen
(70/30%) and maintained with 1.5-2.5% isoflurane during the
entire surgical procedure. Body temperature was monitored
rectally and kept close to 37.5 °C. A catheter was inserted into
the tail vein.
2. In Vitro Stability of [11C]3 in Rat Blood Samples. Com-
pound [11C]3 (10 MBq) formulated in a solution containing
NaCl 0.9%/phosphate buffer 25 mM pH = 7.4/ethanol (v/v/v
80/10/10) was added to blood. After incubation for 5, 15, 30, and
60 min at 37 °C, blood samples (1 mL) were centrifuged (2000g,
4 min). Plasma was mixed with an equivalent volume of
acetonitrile and centrifuged (2000g, 10 min). The radioactivity
of the precipitate was measured to quantify the efficiency of the
acetonitrile extraction. Supernatants were filtered and injected
onto HPLC (Macherey Nagel nucleosil 100-5 protect1 column,
4.6 mm ꢀ 250 mm, 1 mL/min, λ = 254 nm, CH3CN/H2O/Et3N
55:45:0.01).
3. Radioactivity Uptake in Rat Brain. Rats were injected with
[11C]3 (15-120 MBq) formulated in a solution containing NaCl
0.9%/phosphate buffer 25 mM pH = 7.4/ethanol (v/v/v 80/10/
10) and were killed by decapitation at 3, 5, 10, 30, 45, and 60 min
post injection. Whole brain was quickly dissected and rinsed
with saline solution to minimize residual blood. The brain
sample was weighed, and the radioactivity was measured in a
γ-counter (Cobra 2 gamma counter, Packard). Data were
expressed as the percentage of injected dose (decay-corrected)
per gram of tissue (% ID/g).
Figure 4. Radiochromatogram from HPLC analysis after [11C]3
radiosynthesis.
in 95% yield as a yellow powder. 1H NMR (CDCl3): δ 7.70 (s,
1H), 7.66 (d, 1H, J = 8.0 Hz), 7.56 (s, 1H), 7.32 (d, 1H, J = 7.0
Hz), 7.06-7.14 (m, 3H), 6.95 (t, 1H, J = 7.5 Hz), 6.75 (d, 1H,
J = 7.8 Hz), 4.42 (s, 2H), 3.82 (s, 2H), 3.25 (s, 3H). 13C NMR
(CDCl3): δ 156.3, 152.8, 142.3, 136.1, 135.9, 135.2, 129.6, 129.4,
128.9, 128.4, 127.9, 126.1, 125.5, 122.3, 111.7, 93.7, 43.4, 38.0,
30.9.
1-Methylquinolinium-3-carboxylic Acid Trifluromethanesul-
fonate (5). Under nitrogen, 3-quinoline carboxylic acid (173 mg,
1 mmol) was dissolved in acetonitrile (20 mL) at 60 °C. Methyl
trifluoromethane sulfonate (124 μL, 1.1 mmol) was added to the
solution. The mixture was stirred at 60 °C for 2 h. After
evaporation of the solvent, diethyl ether (3 mL) and dichloro-
methane (3 mL) were added, and after 12 h, the precipitate was
collected and dried. Compound 5 was isolated in 45% yield as a
4. [11C]3 Oxidation Rate in Blood and Brain Samples. Rats
were injected with [11C]3 (15-120 MBq) formulated in a solu-
tion containing NaCl 0.9%/phosphate buffer 25 mM pH = 7.4/
ethanol (v/v/v 80/10/10) and were killed by decapitation at 3, 5,
10, 20, 30, 45, and 60 min post injection. Whole brain was
quickly dissected and rinsed with saline solution to minimize
residual blood and intracardiac blood sample was collected.
Brain was crushed (UltraTurrax T25, Janke and Kunkel) in
acetonitrile (2.5 mL) and centrifuged at 2000g for 10 min. The
radioactivity of the precipitate was measured to quantify the
efficiency of the acetonitrile extraction. Supernatants were
filtered and injected onto HPLC (Macherey Nagel nucleo-
sil 100-5 protect1 column 4.6 mm ꢀ 250 mm, 1 mL/min, λ =
254 nm, CH3CN/H2O/Et3N 55:45:0.01). After centrifugation of
blood samples (2000g, 4 min, 1 mL), plasma was mixed with
an equivalent volume of acetonitrile and centrifuged (2000g,
10 min). The radioactivity of the precipitate was measured to
quantify the efficiency of the acetonitrile extraction. Super-
natants were filtered and injected onto HPLC as described
above.
5. LC-MS/MS Analysis. Rats were injected with 3 (1 mg, 2.37
μmol) formulated in a solution containing NaCl 0.9%/phos-
phate buffer 25 mM pH = 7.4/ethanol (v/v/v 80/10/10) and were
killed by decapitation at 30 min post injection. Whole brain was
dissected, crushed (UltraTurrax T25, Janke and Kunkel) in
acetonitrile (2.5 mL), and centrifuged at 2000g for 10 min.
Supernatants were concentrated and analyzed by LC-MS/MS.
Analyses were performed with a HPLC Surveyor chain con-
nected online to an orthogonal electrospray source (Deca XP
MS-n ThermoFinnigan) operated in the positive electrospray
ionization mode (ESIþ). The ions were focused into an ion trap
capable of MS and MS/MS analyses. The mass spectra were
acquired during 35 ms from 100 to 1000 m/z. The capillary exit
of the electrospray ion source was set at 70 V, the octapole at 3 V,
and the capillary temperature at 200 °C. A counter flow of
nitrogen was used as nebulizing gas. Xcalibur data system was
used to acquire the data, which were further processed with a
Turbo Sequest data system. The sample was resuspended in
0.1% acetic acid in water, and 5 μL were injected onto a C18
Thermo electron corporation C18 column (0.5 mm ꢀ 50 mm,
5 μm) that had been equilibrated with 95% mobile phase A
(0.1% aq acetic acid) and 5% mobile phase B (0.1% acetic acid
1
beige solid. H NMR (DMSO-d6): δ 14.0 (s, 1H), 9.96 (s, 1H),
9.83 (s, 1H), 8.57-8.69 (m, 2H), 8.42 (d, 1H), 8.16 (d, 1H), 4.74
(s, 3H). 13C NMR (DMSO-d6): δ 163.5, 150.5, 147.7, 139.3, 137.4,
131.8, 130.5, 128.4, 124.8, 119.3, 45.3.
N-(3-Iodo-benzyl)-N0-(1-[11C]methyl-3-carbonyl-1,4-dihydro-
quinoline)-guanidine ([11C]3). [11C]Methyl triflate was trapped
at room temperature in a solution containing the quinoline 1
(1 mg, 3.1 μmol) in acetonitrile (150 μL).31 When all the radio-
activity was collected, a solution of BNAH (2 mg, 9.3 μmol) in
CH3CN (150 μL) was added to the mixture. The resulting
mixture was kept at room temperature for 5 min. The purifica-
tion was performed by reverse-phase semipreparative HPLC
(Macherey Nagel nucleosil 100-5 protect1 column 10 mm ꢀ
250 mm, 4 mL/min, λ = 254 nm, CH3CN/H2O/Et3N 55:45:0.01)
(Figure 4). The fraction containing the labeled product [11C]3
(tR = 18.5 min) was collected into a flask containing water
(25 mL). The radioactive solution was transferred onto a
Macherey Nagel chromabond C18ec previously conditioned
with methanol (2 mL) and water (5 mL). [11C]3 was eluted with
ethanol (200 μL), and an isotonic saline solution containing
10% phosphate buffer (pH = 7.4 25 mM) was added (1 mL).
The final solution was filtered through a 0.22 μm filter into a
sterile vial. The radiochemical and chemical purities and spe-
cific activities were determined by reverse-phase analytical
HPLC (Macherey Nagel nucleosil 100-5 protect1 column
4.6 mm ꢀ 250 mm, 1 mL/min, λ =254 nm, CH3CN/H2O/
Et3N 45:55:0.01, tR[11C]3 = 13 min). Radiochemical purity of
[11C]3 exceeded 99% and batches of 550-740 MBq were ready
for injection in about 50 min.
Biological Evaluation. 1. Animals. Animal experimental
procedures were in accordance with the recommendations of
the EEC (86/609/EEC) and the French National Committee
(decret 87/848) for the care and use of laboratory animals and
were approved by the Normandy Regional Animal Ethics
Committee (saisine N/05-04-05-05). Sprague-Dawley male rats
weighing 250-300 g were used in all experiments. The animals
were kept at constant temperature (22 °C) and humidity (50%)
with 12 h light/dark cycles and were allowed free access to food

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1287
in acetonitrile). The peaks were eluted with a linear acetonitrile
gradient of 8% per min. A split ratio of 30:1 was used to perfuse
the column at a flow rate of 10 μL/min. The HPLC column was
rinsed with 90% acetonitrile in 0.1% acetic acid between each
injection. The MS data was acquired in scan mode considering
the positive ion signal.
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Acknowledgment. We thank the CEA and the CNRS for
financial and technical support. We thank Ahmed Abbas,
ꢀ ^
Jerome Delamare, and Martine Dhilly for their technical
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Supporting Information Available: Synthesis of compounds 1
and 4. This material is available free of charge via the Internet at
http://pubs.acs.org.
(18) Foucout, L.; Gourand, F.; Dhilly, M.; Bohn, P.; Dupas, G.;
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