Facile synthesis and PET imaging of a novel potential heart acetylcholinesterase tracer N-[11C]methyl-3-[[(dimethylamino) carbonyl]oxy]-2-(2′,2′-diphenylpropionoxymethyl)pyridinium

Article abstract of DOI:10.1016/j.bmcl.2005.07.034

A new AChE tracer N-[¹¹C]methyl-3-[[(dimethylamino)carbonyl]oxy] -2-(2′,2′-diphenylpropionoxymethyl)pyridinium ([¹¹C]MDDP, [¹¹C]1) has been synthesized in 40-65% radiochemical yield. Initial PET dynamic studies of [¹¹

Full text of DOI:10.1016/j.bmcl.2005.07.034

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4510–4514
Facile synthesis and PET imaging of a novel potential
heart acetylcholinesterase tracer N-[ C]methyl-3-
1
1
0
0
[
[(dimethylamino)carbonyl]oxy]-2-(2 ,2 -
diphenylpropionoxymethyl)pyridinium
a
a
a
Ji-Quan Wang, Michael A. Miller, Bruce H. Mock, John C. Lopshire,
b
b
William J. Groh, Douglas P. Zipes, Gary D. Hutchins and Qi-Huang Zheng
b
a
a,
*
a
Department of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Department of Medicine, Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
b
Received 15 June 2005; revised 5 July 2005; accepted 6 July 2005
Available online 19 August 2005
1
Abstract—A new AChE tracer N-[ C]methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2 ,2 -diphenylpropionoxymethyl)pyridinium
1
0
0
1
[ C]MDDP, [ C]1) has been synthesized in 40–65% radiochemical yield. Initial PET dynamic studies of [ C]MDDP in rat heart
1
11
11
(
showed rapid heart uptake and blood pool clearance to give high-quality heart images. Blocking studies of [ C]MDDP with pre-
1
1
1
treatment drug neostigmine in rats found only minor reductions in rat heart [ C]MDDP retention. The results suggest that
1
1
1
11
C]MDDP delineates the heart very clearly, and the uptakes of [ C]MDDP in rat heart might be related to non-specific binding.
2005 Elsevier Ltd. All rights reserved.
[
ꢀ
The function of acetylcholinesterase (AChE) is to termi-
were designed, synthesized, and evaluated as therapeutic
2
nate nerve impulse transmission by hydrolyzing the neu-
rotransmitter acetylcholine. There is overwhelming
consensus that acute exposure to organophosphorus
drugs by Leader et al. The results show that the com-
pound N-methyl-3-[[(dimethylamino)carbonyl]oxy]-2-
0
0
(2 ,2 -diphenylpropionoxymethyl)pyridinium (MDDP)
iodide inhibited AChE selectively over BChE, with a
bimolecular rate constant similar to that of pyridostig-
mine. Our objective is to develop heart AChE tracers
for biomedical imaging of cardiac neurotransmission
(
OP) agents inhibits AChE and that toxicity and lethal-
1
ity arise due to inhibition of AChE. The protection
against OP poisoning in current prophylactic and thera-
peutic regimens is a pretreatment regimen consisting of
pyridostigmine, the reversible, covalent AChE inhibitor;
the muscarinic receptor antagonist atropine (ATR); and
the AChE reactivator pralidoxime chloride. Additional
treatment with diazepam has proven advantageous in
attempts to prevent convulsions. These regimens pro-
vide extensive protection against OP intoxication in
animals. However, treatment might be considerably
simplified if drugs that possessed multiple protective
functions were used. To test this hypothesis, a group
of pyridophen analogues, binary pyridostigmine-apro-
phen prodrugs with differential inhibition of AChE,
butyrylcholinesterase (BChE), and muscarinic receptors,
3
using positron emission tomography (PET). Many pos-
itron labeled AChE inhibitors have been developed and
evaluated for the in vivo mapping of AChE. Radiotra-
cers with a low selectivity of AChE over BChE, as well
as moderate binding properties to AChE, have led to
non-specific binding in AChE enzyme over-expressed
4
areas, such as brain and heart regions. It was expected
that this problem could be overcome using radiolabeled
pyridophen analogues with an excellent anti-AChE
activity and a high selectivity of AChE over BChE.
1
1
5
We have previously developed [ C]neostigmine,
1
1
6
[ C]edrophonium, and their analogues as potential
PET heart imaging agents for AChE. These compounds
exhibited either high non-specific accumulation in myo-
cardial tissue or poor myocardial uptake and were con-
sidered unsuitable for imaging the vagal system of the
heart. As part of this project we turned our efforts
1
1
0
0
Keywords: N-[ C]methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2 ,2 -
diphenylpropionoxymethyl)pyridinium; Tracer; Synthesis; Positron
emission tomography; Acetylcholinesterase; Heart.
*
Corresponding author. Tel.: +1 317 278 4671; fax: +1 317 278
711; e-mail: qzheng@iupui.edu
1
1
toward the development of [ C]pyridostigmine and its
9
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.034

J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4510–4514
4511
1
labeled analogues ([ C]para-pyridostigmine and [ C]-
1
11
major product in 31% chemical yield from the carba-
mylation of 3-hydroxy-2-hydroxymethylpyridine HCl
(2) with dimethylcarbamyl chloride. The other two
products isolated from the reaction mixture were
found to be the isomeric carbamate and the bis-carba-
mate. The acylation agent 2,2-diphenylpropionyl chlo-
ride (5) was prepared from 2,2-diphenylpropionic acid
(4) with thionyl chloride in 96% yield. Acylation of 3
with 5 afforded the carbamyl-ester precursor 3-[[(di-
7
ortho-pyridostigmine) (Fig. 1). In this ongoing study,
we investigated whether N-[ C]methyl-3-[[(dimethylami-
1
1
0
0
no)carbonyl]oxy]-2-(2 ,2 -diphenylpropionoxymethyl)pyrid-
1
1
11
inium ([ C]MDDP, [ C]1) could be used to map heart
AChE in vivo. As part of our efforts to evaluate novel
potential heart imaging agents, we synthesized the tracer
[ C]MDDP and performed initial PET imaging studies
of the tracer [ C]MDDP in rat heart.
1
1
1
1
0
0
methylamino)carbonyl]oxy]-2-(2 ,2 -diphenylpropionoxy-
methyl)pyridine (6) in 51% yield. The tertiary pyridine
6 was methylated with methyl triflate to give the dimeth-
1
1
The synthetic approach for the tracer [ C]MDDP is
shown in Scheme 1. The synthesis of precursor and
reference standard was performed using a modification
0
0
ylamino)carbonyl]oxy]-2-(2 ,2 -diphenylpropionoxymeth-
yl)pyridinium triflate (1) in 97% yield. A simple
2
of the literature procedure. The key intermediate,
2
5
–9
-hydroxymethyl-3-dimethylaminocarbonyl-oxypyridine
3), was isolated by column chromatography as the
technique solid-phase extraction (SPE) for conve-
nient labeling and isolation of [ C-methyl]quaternary
1
1
(
O
CH
3
OH
O
N
CH
3
11CH
OTf
CH CH
3
N
N
3
H C
OTf
11CH
2
3
CH
3
3
CH
3
[11
C]Neostigmine
[11C]Edrophonium
O
CH
3
O
N
O
O
CH
3
O
N
CH
3
N
CH
3
H
3
C
O
OTf
N
N
N
OTf
3
OTf
3
11CH
11CH
11CH
3
CH3
[11
C]Pyridostigmine
[11C]para-Pyridostigmine
[11C]ortho-Pyridostigmine
1
1
11
Figure 1. Chemical structures of [ C]neostigmine, [ C]edrophonium, [ C]pyridostigmine, [ C]para-pyridostigmine, and [ C]ortho-pyridostigmine.
11
11
11
(
CH
3 2
) NOCO
OH
3 2
OCON(CH )
(
CH
3
2
) NCOCl
CH
3
pyridine
COOCH
2
N
CH
2
OH
CH3
COCl
N
N
3
2
CH OH
2
SOCl
2
CH
3
6
COOH
CH
3
OTf
5
3 2
(CH ) NOCO
CH
3
4
COOCH
2
N
OTf
CH
3
(
CH
3 2
) NOCO
CH
3
11CH
OTf, CH CN
3 3
1
COOCH
2
6
N
OTf
3
11CH
[11C]MDDP, [11C]1
1
1
0
0
11
Scheme 1. Synthesis of N-[ C]methyl-3-[[(dimethylamino)carbonyl]oxy]-2-(2 ,2 -diphenylpropionoxymethyl)pyridinium ([ C]MDDP, [ C]1).
11

4512
J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4510–4514
1
amines by N-[ C]methylation method was employed
1
1
in the radiosynthesis of target tracer [ C]MDDP
1
1
[ C]1). The tertiary pyridine precursor 6 was labeled
1
(
with [ C]methyl triflate to provide quaternary pyridi-
1
1
1
1
nium tracer [ C]1 in 40–65% radiochemical yields, de-
cay corrected to end of bombardment (EOB), and a
synthesis time of 10–15 min. The key part in this tech-
nique is a SiO Sep-Pak cartridge containing 0.5–2 g
2
of adsorbent. It can be used to remove unreacted ter-
tiary amine precursor 6, reaction solvent acetonitrile,
1
1
and unreacted [ C]methyl triflate, which was decom-
posed by eluant ethanol. The final labeled product
1
1
11
C-methyl]quaternary amine [ C]1 was eluted with
[
an aqueous solution of 2% acetic acid, which can also
contain up to 8% ethanol to enhance recovery of some
1
1
[
C-methyl]quaternary cations. The SPE technique
was used for fast, efficient preparative separation of
labeled product from its unlabeled precursor with
large polarity difference, which shortened total synthe-
sis and formulation time and afforded higher overall
radiochemical yields. Chemical purity, radiochemical
purity, and specific radioactivity were determined by
the analytical HPLC method. The chemical purities
of the precursor 6 and the reference standard 1 were
1
1
Figure 2. PET images of the tracer [ C]MDDP in female rats
anesthetized with acepromazine (0.2 mg/kg, im) and torbugesic
(0.2 mg/kg, im), administered with 0.2 mCi radioactivity, and scanned
with IndyPET-II for 60 min. The images are coronal slices from scans
of three rats. The upper three panels show images after injection of
11
C]MDDP without a blocking agent. Image intensity is SUV
[
averaged from 20 to 60 min after tracer injection. The lower three
panels show images of the same three rats following administration of
neostigmine as described in the text. The color bar indicates approx-
imate SUV for all six images.
>
95%. The radiochemical purity of the target tracer
1
1
[ C]1 was >99%, and the chemical purity of the tar-
get tracer [ C]1 was >93%. The specific radioactivity
1
1
1
1
of the tracer [ C]1 was 1.0–1.5 Ci/lmol at end-of-syn-
thesis (EOS).
sity is SUV averaged from 20 to 60 min after tracer
injection, and the color bar indicates approximate
SUV for all three images. All PET images of the tracer
1
0
In vivo dynamic PET imaging studies of the tracer
1
1
11
[
rats were performed in an IndyPET-II scanner
C]MDDP in young adult female Sprague–Dawley
1
[ C]MDDP in rats 1, 2, and 3 showed that the heart
1,12
for
0 min post iv injection of 0.2 mCi of the tracer in a
was visible in blocking studies. A color scale normalizes
all sets of data including unblocked control study and
blocked with neostigmine study. Comparing the drug
study with the control study, the partial blocking effect
can be seen from the images.
6
rat, and the heart images in three rats are shown in
the upper three panels in Figure 2. All PET images are
coronal views. The image intensity is standard uptake
value (SUV), averaged from 20 to 60 min after tracer
injection. The color bar indicates approximate SUV
for all three images. All PET images of the tracer
1
1
The dynamic PET data of the tracer [ C]MDDP with
no blocker and with blocker neostigmine are shown in
Figure 3. The plot Figure 3 is the individualized tracer
heart uptake (y-axis) versus time from injection (x-axis),
1
1
[
C]MDDP in rats 1, 2, and 3 showed that the heart
was visible with the tracer. These images indicate that
the new tracer delineates the heart very clearly. All imag-
es were acquired in list-mode and sorted into 15 · 20 s
frames, 10 · 60 s frames, and 9 · 300 s frames. Images
were reconstructed using filtered back projection with
1
1
which indicated the kinetics of the tracer [ C]MDDP
with no blocker and with blocker neostigmine in three
rats. The tracer uptake is expressed as the values, which
are approximate SUV in the cardiac region of interest
(ROI) plotted versus each time point in 60 min of entire
scan time. The initial peak is the first pass of the tracer
following the tail vein injection. The open symbols are
values with no blocking agent in rats 1, 2, and 3 (control
group). The filled symbols are values from scans, follow-
ing the administration of the blocking agent neostigmine
in the same three rats (blocking group). The tracer up-
take was changed in the blocking study, which indicates
a blocking drug effect on the binding of the radiotracer.
ꢀ1
a 70% Hanning filter (4.242 cm cutoff frequency).
In vivo competitive inhibition studies were performed to
1
1
assess the in vivo specificity of the tracer [ C]MDDP to
heart tissue AChE and whether the tracer distribution is
susceptible to indirect pharmacological or pharmacody-
namic effects that could complicate uptake site measure-
ments. Competition or ‘‘blocking’’ study was carried out
by pretreating groups of animals with drugs, unlabeled
AChE inhibitors, which compete, or are believed to
compete for the same binding site that the tracer inter-
The mean and standard deviation (SD) SUV for heart
ROIs of three rats in two different experimental groups
(control group and blocking group) are given in Table 1,
which shows the comparison of average tracer
[ C]MDDP uptake in the heart with no blocker and
with blocker neostigmine in three rats. Likewise, Table
1 suggests that the partial blocking effect appeared in
1
0
acts within the body. For the blocking imaging studies,
the same three rats were pretreated by an intravenous
injection of neostigmine prior to intravenous injection
1
1
11
of the tracer [ C]MDDP. The heart images in three rats
are shown in the lower three panels in Figure 2. Like-
wise, all PET images are coronal views, the image inten-

J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4510–4514
4513
1
1
[
C]MDDP retention after administration of neostig-
rat 1, control
rat 2, control
rat 3, control
rat 1, neostigmine
rat 2, neostigmine
rat 3, neostigmine
mine, the possibility that what we describe as non-specif-
ic binding may actually be insufficient blockade of
cholinesterase binding sites.
The experimental details are given for the new com-
1
1
pound 1 and new tracer [ C]1, and only characteriza-
tion data are given for other known compounds 3, 5,
1
3
and 6. The experimental details for PET imaging stud-
1
ies are also given.
4
In summary, the synthetic procedures that provide ter-
tiary pyridine precursor 6, quaternary pyridinium refer-
ence standard 1, and quaternary pyridinium target
0
10
20
30
40
50
60
1
1
tracer [ C]1 have been well-developed. Initial PET
1
1
Time [min]
dynamic studies of the tracer [ C]1 in rat heart showed
rapid heart uptake and blood pool clearance to give
high-quality heart images. However, the results from
11
Figure 3. Kinetics of [ C]MDDP in three rats. Values are approxi-
mate SUV in cardiac ROI plotted versus time from the beginning of
scan. The initial peak is the first pass of the tracer, following the tail
vein injection. The open symbols are values with no blocking agent.
The filled symbols are values from scans, following the administration
of neostigmine, as described in the text.
1
1
the blocking studies of [ C]MDDP with pretreatment
drug neostigmine showed that no specific binding is
present. These results suggest that the new tracer delin-
eates the heart very clearly, the localization of
1
1
[
C]MDDP in rat heart might be mediated either by
non-specific processes or by insufficient blockade of cho-
linesterase binding sites, and the visualization of
[ C]MDDP-PET on rat heart might be associated with
non-specific binding or insufficient blockade of cholines-
terase binding sites.
Table 1. Mean and standard deviation (SD) SUV for heart ROIs of
three rats
1
1
Experimental group
Mean
SD
Control
Blocking with neostigmine
p value
0.125
0.128
0.87
0.034
0.066
Acknowledgments
the blocking study. A t test comparing the means of the
This work was partially supported by the Donald W.
Reynolds Foundation and the Indiana Genomics Initia-
tive (INGEN) of Indiana University, which is supported
in part by Lilly Endowment Inc. The authors thank
Winston Baity, Terry McBride, Tanya Martinez, and
Kristan Boling for their assistance in animal studies.
The refereesÕ criticisms and editorÕs comments for the
revision of the manuscript are greatly appreciated.
1
1
tracer [ C]MDDP with no blocker and with blocker
neostigmine gave a p value of 0.87, which did not denote
the presence of a statistically significant difference. Ini-
1
1
tial PET blocking studies of the tracer [ C]MDDP with
pretreatment drug neostigmine in rats found only minor
1
1
reductions in rat heart [ C]MDDP retention. Therefore,
we conclude there was no significant blocking in the
1
1
data set and the uptakes of the tracer [ C]MDDP in
rat heart might be the results of non-specific binding.
Based on the data available from the in vivo PET stud-
ies, we can assume that while in vitro experiments indi-
cate efficacy of an AChE inhibitor MDDP iodide,
kinetic factors, and rapid blood clearance make
References and notes
1. Duysen, E. G.; Li, B.; Xie, W.; Schopfer, L. M.;
Anderson, R. S.; Broomfield, C. A.; Lockridge, O. J.
Pharmacol. Exp. Ther. 2001, 299, 528.
. Leader, H.; Wolfe, A. D.; Chiang, P. K.; Gordon, R. K.
J. Med. Chem. 2002, 45, 902.
. Carrio, I. J. Nucl. Med. 2001, 42, 1062.
. Ryu, E. K.; Choe, Y. S.; Park, E. Y.; Paik, J-Y.; Kim, Y.
R.; Lee, K-H.; Choi, Y.; Kim, S. E.; Kim, B-T. Nucl. Med.
Biol. 2005, 32, 185.
1
1
[
C]MDDP unsuitable as tracers for PET imaging of
2
AChE enzyme in the heart. This work demonstrates that
the therapeutic drug and imaging tracer have different
pharmacokinetic profiles. Further studies will determine
whether the uptake site is AChE or if other binding sites
are also involved.
3
4
5
6
7
8
. Mulholland, G. K.; Zheng, Q.-H.; Mock, B. H.; Carlson,
K. A.; Giger, S.; Vavrek, M. T.; Fain, R. L.; Hutchins, G.
D. J. Nucl. Med. 1999, 40(Suppl. 5), 495.
. Zheng, Q.-H.; Liu, X.; Fei, X.; Wang, J.-Q.; Mock, B. H.;
Glick-Wilson, B. E.; Sullivan, M. L.; Hutchins, G. D.
Bioorg. Med. Chem. Lett. 2003, 13, 1787.
. Wang, J.-Q.; Miller, M. A.; Fei, X.; Stone, K. L.;
Lopshire, J. C.; Groh, W. J.; Zipes, D. P.; Hutchins, G.
D.; Zheng, Q.-H. Nucl. Med. Biol. 2004, 31, 957.
. Mulholland, K. M.; Zheng, Q.-H.; Mock, B. H.; Vavrek,
M. T. J. Labelled Cpd. Radiopharm. 1999, 42(Suppl. 1),
S459.
Because of the high concentrations of AChE and
BuChE in blood, as well as in heart and brain, it is
extremely difficult to measure the blockade of cholines-
terase function in these latter tissues. Typically, there is a
narrow window between measurable blockade and
lethality. In fact, it has been shown that blockade of
blood AChE binding sites with neostigmine can increase
the amount of AChE radiotracers available for binding
in brain and likely heart as well. As 2 out of 3 of the rats
used in this study actually exhibited increases in

4
514
J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4510–4514
CH Cl (10 mL) at room temperature. The solution was
9
. Zheng, Q.-H.; Stone, K. L.; Mock, B. H.; Miller, K. D.;
Fei, X.; Liu, X.; Wang, J.-Q.; Glick-Wilson, B. E.; Sledge,
G. W.; Hutchins, G. D. Nucl. Med. Biol. 2002, 29, 803.
2
2
stirred for 30 min. TLC showed no starting material left.
The solvent was removed under vacuum to give com-
1
1
0. Zheng, Q.-H.; Fei, X.; DeGrado, T. R.; Wang, J.-Q.; Stone,
K. L.; Martinez, T. D.; Gay, D. J.; Baity, W. L.; Mock, B.
H.; Glick-Wilson, B. E.; Sullivan, M. L.; Miller, K. D.;
Sledge, G. W.; Hutchins, G. D. Nucl. Med. Biol. 2003, 30,
53.
1. Rouze, N. C.; Hutchins, G. D. IEEE Trans. Nucl. Sci.
003, 50, 1491.
2. Frese, T.; Rouze, N. C.; Bouman, C. A.; Sauer, K.;
Hutchins, G. D. IEEE Trans. Med. Imaging 2003, 22, 258.
3. Experimental details and characterization data. (a) Gen-
eral: All commercial reagents and solvents were used
without further purification unless otherwise specified.
Melting points were determined with a MEL-TEMP II
pound 1 (0.56 g, 97%) as a white solid, mp 43–45 ꢁC. H
3
NMR (CDCl , 300 MHz) d 8.64 (d, 1H, J = 6.6 Hz,
pyridyl H), 8.34 (d, 1H, J = 8.8 Hz, pyridyl H), 7.90 (dd,
1H, J = 8.9 Hz, J = 5.9 Hz, pyridyl H), 7.02-7.30 (m,
1
2
+
7
10H, phenyl H), 5.58 (s, 2H, CH
3.11 (s, 3H, NCH ), 3.01 (s, 3H, NCH
2
), 4.08 (s, 3H, N CH
), 1.90 (s, 3H, CH
3
),
).
1
1
1
3
3
3
+
2
LRMS (EI, m/z): 181 (100%), 419 [(MꢀOTf) , 1.8%].
27 4 2
HRMS (EI, m/z): calcd for C25H O N : 419.1970, found:
1
1
419.1971. (f) Tracer [ C]1: The precursor (6, 0.5 mg) was
dissolved in acetonitrile (250 lL). The mixture was trans-
ferred to a small volume, three-necked reaction tube.
1
1
[ C]Methyl triflate was passed into an air-cooled reaction
tube at a temperature between ꢀ15 and ꢀ20 ꢁC, which
was generated by a Venturi cooling device powered with
100 psi compressed air, until radioactivity in solution
reached a maximum (2–3 min), and then the reaction tube
was isolated and heated at 70–80 ꢁC for 2–3 min. The
reaction tube was connected to the Silica Sep-Pak. The
labeled product solution mixture was passed onto the SiO2
Sep-Pak for SPE purification by gas pressure. The reaction
tube and Sep-Pak were washed with ethanol (5 mL), and
the washing solution was discarded to a waste bottle. The
1
capillary tube apparatus and were uncorrected. H NMR
spectra were recorded on a Bruker QE 300 NMR
spectrometer using tetramethylsilane (TMS) as an internal
standard. Chemical shift data for the proton resonances
were reported in parts per million (d), relative to the
internal standard TMS (d 0.0). Low-resolution mass
spectra (LRMS) were obtained using a Bruker Biflex III
MALDI-Tof mass spectrometer, and high-resolution mass
spectra (HRMS) were obtained using a Kratos MS80 mass
spectrometer, in the Department of Chemistry at Indiana
University. Chromatographic solvent proportions are
expressed on a volume: volume basis. Thin layer chroma-
1
1
final product [ C]1 was eluted from the Sep-Pak with
90:8:2 H O/EtOH/HOAc (2–4 mL), sterile-filtered through
2
a 0.22 lm cellulose acetate membrane, and collected into a
sterile vial. The pH was adjusted to 5.5–7.0 with 2 M
NaOH and 150 mM NaH PO mixed solution (1/20, 0.2–
2 4
tography was run using Analtech silica gel GF uniplates
(
2
5 · 10 cm ). Plates were visualized by UV light. Normal
phase flash chromatography was carried out on EM
Science silica gel 60 (230–400 mesh) with a forced flow of
the indicated solvent system in the proportions described
below. All moisture-sensitive reactions were performed
under a positive pressure of nitrogen maintained by a
direct line from a nitrogen source. Analytical HPLC was
performed using a Prodigy (Phenomenex) 5 lm C-18
0.4 mL). Total radioactivity was assayed and the total
volume (2.5–5.0 mL) was noted. The overall synthesis time
1
1
was 10–15 min. The decay corrected yields, from CO2,
were 40–65%, and the radiochemical purity was >99% by
analytical HPLC. Retention times in the analytical HPLC
1
1
system were: RT6 = 4.51 min, RT[ C]1 = 2.83 min. The
chemical purity of the target tracer was >93%.
1
1
column, 4.6 · 250 mm; 3:1:1 CH
3
CN/MeOH/20 mM, pH
(buffer solution) mobile phase; flow rate
14. (a) Dynamic IndyPET-II imaging of the tracer [ C]MDDP
in the rats. All animal experiments were performed under a
protocol approved by the Indiana University Institutional
Animal Care and Use Committee. The female Sprague–
Dawley rat (250–300 g) was anesthetized with aceproma-
zine (0.2 mg/kg, im) and torbugesic (0.2 mg/kg, im). 0.2 mCi
ꢀ
6
1
.7 KHPO
4
.5 mL/min; and UV (240 nm) and c-ray (NaI) flow
Sep-Pak type cartridge was
obtained from Waters Corporate Headquarters, Milford,
MA. Sterile Millex-GS 0.22 lm vented filter unit was
obtained from Millipore Corporation, Bedford, MA. (b)
= 0.13 (50:1
, 300 MHz) d 8.42 (d,
H, J = 3.7 Hz, ring-H), 7.55 (dd, 1H, J = 8.1 Hz,
detectors. Semi-prep SiO
2
1
1
of [ C]MDDP was administered intravenously to the rat
via the tail vein. The micro-PET images of the tracer were
acquired in IndyPET-II scanner by the ordered subset
expectation maximization (OSEM) using 6 subsets/4 itera-
tions for 60 min dynamic scans from a rat post intravenous
injection of 0.2 mCi of the tracer, and frame durations were
defined as 300 s for entire 3600 s scan. (b) Blocking
Compound 3: a white solid, yield 31%, R
f
1
2 2 3
CH Cl /MeOH). H NMR (CDCl
1
1
J2 = 1.5 Hz, ring-H), 7.27 (dd, 1H, J1 = 8.1 Hz,
J
2
= 5.1 Hz, ring-H), 4.73 (s, 2H, CH
2
OH), 4.22 (br s,
). (c)
1
H, OH), 3.13 (s, 3H, CH ), 3.03 (s, 3H, CH
3
3
1
11
Compound 5: a white solid, yield 96%, mp 35–37 ꢁC. H
NMR (CDCl , 300 MHz) d 7.18–7.42 (m, 10H, phenyl H),
.09 (s, 3H, CH ). (d) Compound 6: a colorless oil, yield
1%, R = 0.48 (1:1 EtOAc/hexane). H NMR (CDCl
IndyPET-II imaging of the tracer [ C]MDDP in the rats
3
with pretreatment drug neostigmine. For the blocking
experiments, the rats (250–300 g) were pretreated by an
intravenous injection in the tail vein with 3.0 mg/kg of
neostigmine in saline 30 min, prior to iv injection of 0.2 mCi
2
5
3
3
1
f
3
,
00 MHz) d 8.42 (dd, 1H, J = 4.4 Hz, J = 1.5 Hz, pyridyl
1
2
1
1
H), 7.56 (dd, 1H, J
(
1
= 8.1 Hz, J
2
= 1.5 Hz, pyridyl H), 7.30
2
= 5.2 Hz, pyridyl H), 7.18–7.25 (m,
[ C]MDDP in the tail vein. After tracer administration, the
animals were handled, as described above. (c) Statistical
analysis. Differences between neostigmine pretreated group
and control group were examined for statistical significance
using StudentÕs t test. A p value less than 0.05 denoted the
presence of a statistically significant difference.
dd, 1H,J
1
= 8.1 Hz, J
1
2
0H, phenyl H), 5.31 (s, 2H, CH ), 2.94 (s, 3H, CH N),
3
2
.87 (s, 3H, CH N), 1.92 (s, 3H, CH ). (e) Compound 1:
3
3
Methyl triflate (115 lL, 1.016 mmol) was added to a
solution of compound 6 (0.41 g, 1.014 mmol) in dry