11C-Fallypride: Radiosynthesis and preliminary evaluation of a novel dopamine D2/D3 receptor PET radiotracer in non-human primate brain

Article abstract of DOI:10.1016/j.bmc.2003.10.020

Fallypride [benzamide, 5-(3-fluoropropyl)-2,3-dimethoxy-N-[(2S)-1-(2- propenyl)-2-pyrrolidinyl]methyl]-, CAS RN 166173-78-0] is a selective dopamine D2/D3 receptor antagonist. Carbon-11 labeled fallypride may serve as a radiotracer for use in biomedical imaging technique positron emission tomography (PET). The precursor, 5-(3-fluoropropyl)-2-hydroxy-3-methoxy-N-[(2S)- 1-(2-propenyl)-2-pyrrolidinyl]methyl]benzamide was synthesized from 2-hydroxy-3-methoxy-5-(2-propenyl)benzoic acid, methyl ester in seven steps with approximately 10% overall chemical yield. Using this precursor ¹¹C-fallypride was produced by radiolabeling with ¹¹C-methyl iodide in 25-40% radiochemical yields with specific activities of 200-1000 Ci/mmol. PET imaging studies in nonhuman primates with ¹¹C-fallypride showed radiotracer localization in dopaminergic brain regions such as caudate, putamen, thalamus and cortex. This regional localization of ¹¹C-fallypride is similar to that observed previously for ¹⁸F-fallypride. The results suggest ¹¹C-fallypride is a useful PET radiotracer for imaging dopamine D2/ D3 receptors.

Full text of DOI:10.1016/j.bmc.2003.10.020

Bioorganic & Medicinal Chemistry 12 (2004) 95–102
11C-Fallypride: radiosynthesis and preliminary evaluation of a
novel dopamine D2/D3 receptor PET radiotracer in non-human
primate brain§
Jogeshwar Mukherjee,a,* Bingzhi Shi,b Bradley T. Christian,b Sankha Chattopadhyaya
and Tanjore K. Narayananb
aBrain Imaging Center, Department of Psychiatry and Human Behavior, University of California-Irvine, Irvine, CA 92697, USA
bDepartment of Nuclear Medicine, Kettering Medical Center, Wright State University, Dayton, OH 45429, USA
Received 30 August 2003; revised 9 October 2003; accepted 10 October 2003
Abstract—Fallypride [benzamide, 5-(3-fluoropropyl)-2,3-dimethoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]-, CAS RN
166173-78-0] is a selective dopamine D2/D3 receptor antagonist. Carbon-11 labeled fallypride may serve as a radiotracer for use in
biomedical imaging technique positron emission tomography (PET). The precursor, 5-(3-fluoropropyl)-2-hydroxy-3-methoxy-N-
[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]benzamide was synthesized from 2-hydroxy-3-methoxy-5-(2-propenyl)benzoic acid,
methyl ester in seven steps with approximately 10% overall chemical yield. Using this precursor 11C-fallypride was produced by
radiolabeling with 11C-methyl iodide in 25–40% radiochemical yields with specific activities of 200–1000 Ci/mmol. PET imaging
studies in nonhuman primates with 11C-fallypride showed radiotracer localization in dopaminergic brain regions such as caudate,
putamen, thalamus and cortex. This regional localization of 11C-fallypride is similar to that observed previously for 18F-fallypride.
The results suggest 11C-fallypride is a useful PET radiotracer for imaging dopamine D2/D3 receptors.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
value when pharmacological or behavioral challenges
are being studied. It can avoid movement of the
subject from the PET scanner. Also, if drug effects
are being explored, then repeat studies can be per-
formed within 2–3 h of the first study. This is not
the case of fluorine-18 labeled radiotracers. It will
generally take an overnight wait to repeat a study
with fluorine-18. Carbon-11 radiotracers allow good
imaging statistics only for about an hour to 90 min.
Thus, the radiotracer ought to achieve optimal bind-
ing kinetics within this time period. In the case of
fallypride, optimal binding kinetics may be achieved
in the extrastriatal regions (such as thalamus, amyg-
dala and cortical regions) in 90 min, but not in the
striatal regions (caudate, putamen and ventral stria-
tum). We have previously shown that amphetamine-
induced dopamine release is able to compete with the
binding of 18F-fallypride in both striatal and extra-
striatal brain regions.3,4 We thus postulated that there
may be a potential use of 11C-fallypride in same-day
repeat studies of extrastriatal brain regions. This will
allow the study of changes in 11C-fallypride binding
resulting from pharmacological or behavioral
challenges.
Fallypride [benzamide, 5-(3-fluoropropyl)-2,3-dime-
thoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]-,
CAS RN 166173-78-0; Fig. 1 1a] is a well-known dopa-
mine D2/D3 antagonist. Fallypride radiolabeled with
fluorine-18 (Fig. 1 1b) is currently being used as a
dopamine D2/D3 receptor imaging agent in positron
emission tomographic (PET) studies.1 The high affinity
(Ki=30 pM for D2 receptor sites), selectivity and rapid
clearance from nonspecific binding sites enables visuali-
zation of a range of receptor concentrations—from very
small levels in the cortex to the very high levels in the
putamen. This has enabled human and non-human
quantitative PET studies using fluorine-18 labeled
fallypride.1,2
Carbon-11 labeled radiotracers have a potential advan-
tage of back-to-back same-day studies. This can be of
§Presented in part at the Annual Society of Nuclear Medicine Meet-
ing, June 22–25, 2003, New Orleans, LA, USA.
* Corresponding author. Tel.: +1-949-824-2018; fax: +1-949-824-
7873; e-mail: mukherjj@uci.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.020

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J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
Fallypride is a substituted benzamide that contains a
dimethoxy substituent group in the benzamide ring. The
2-methoxy group is a common feature in this class of
compounds which can be replaced with a carbon-11
label. This has been done with 11C-raclopride and 11C-
FLB 457 successfully.5,6 Other potential sites on fally-
pride for introduction of a carbon-11 label include the
3-methoxy group, the N-allyl group and the C-fluoro-
alkyl group. Incorporation of a carbon-11 label in the
latter two positions will be more difficult in comparision
with that of the methoxy group. Radiolabeling of the 2-
methoxy group was considered more viable than the 3-
methoxy group because of synthetic ease in obtaining
the phenolic precursor. We therefore decided to intro-
duce carbon-11 label as a methyl group in the methoxy
group at the 2-position (Fig. 1, 1c) to make 11C-fally-
pride. We report here the radiosynthesis of 11C-fally-
pride and preliminary 11C-fallypride-PET imaging
studies in a non-human primate.
base as reported previously.8 In order to convert the
alcohol to the corresponding fluoride, either treatment
with diethylaminosulfur trifluoride (DAST) or conver-
sion to the tosylate followed by treatment with tetra-
butylammonium fluoride (TBAF) may be possible and
has been reported by us and others.7,8 Here, we con-
verted the alcohol to the tosylate and subsequently
reacted with TBAF in order to obtain the corresponding
fluoride 6. These reactions proceeded in moderate to
high yields.
Saponification of the ester group yielded the acid 7. This
acid was then used to couple to the amine 8 in an
amide linkage. The amide could be formed by a variety
of procedures. We used DAST to convert the acid 7 to
the corresponding acid fluoride which was then coupled
to the amine. This proceeded smoothly and gave high
yields similar to what has been reported for related
compounds.9 Deprotection of the MEM group using
titanium chloride provided the final product 10 in good
yields. However, titanium chloride also resulted in
exchanging with the fluorine to provide the corre-
sponding chlorinated analogue (approximately 30–40%
of the product was chlorinated). This created a problem
due to the inability to completely remove the chlori-
nated product from the desired fluorinated product 10.
Alternatively, the deprotection of the MEM group was
carried out by using trifluoroacetic acid (TFA). As
shown in Figure 2, the MEM protected acid was treated
with TFA to provide the deprotected phenol 11. This
was then directly coupled with the amine 8 by the use of
benzotriazol-1-yloxy-tris(dimethylamino)phosphonium
hexafluorophosphate (BOP). This avoided formation of
the chlorinated side-product and the desired product 10
was obtained in approximately 50% yield. Unlabeled
fallypride was synthesized as reported previously.8
2. Results and discussion
2.1. Chemistry
In order to synthesize 11C-fallypride by O-11C-methyl-
ation, a precursor containing a free phenolic group had
to be synthesized. Although either of the two O-methyl
groups found in fallypride could be demethylated using
boron tribromide or other demethylating agents, the
carbon–fluorine bond was found to be unstable, result-
ing in the loss of fluorine (Mukherjee et al., unpublished
observations). Therefore, an alternate, longer synthesis
route of the precursor containing the phenolic group
was essential in order to ensure stability of the molecule.
The precursor 5-(3-fluoropropyl)-2-hydroxy-3-methoxy-
N-[(2S)-1-(2-propenyl)-2-pyrrolidinyl]methyl]benzamide
(10) was synthesized as shown in Figure 2. The starting
material 5-allyl-3-methoxysalicylic acid, methyl ester 2
was prepared in three steps from 3-methoxysalicylic acid
using reported procedures.7 The phenolic group at the
2-position was protected using a 2-methoxyethoxy-
methyl (MEM) ether group. The allyl group was con-
verted to the corresponding alcohol 4 by treatment with
dihydroborane followed with hydrogen peroxide and
2.2. Radiochemistry
Radiolabeling of the phenolic precursor was carried out
by using methods reported previously for preparing 11C-
raclopride and 11C-FLB 457.5,6 Carbon-11 methyl
iodide was efficiently prepared by delivering 11C-CO2
from the cyclotron to the molecular sieve trap in the
General Electric methyl iodide unit (GE box; Fig. 3a).
The trapped 11C-CO2 was then reduced to 11C-CH4 and
eventually to 11C-CH3I in the GE box. We have pre-
viously used this approach for the production of 11C-
methionine, 11C-choline and 11C-flumazenil all of which
require 11C-methylation.10 The conversion of 11C-CO2
to 11C-CH4 avoids the use of lithium aluminum hydride
(LAH; converts 11C-CO2 to 11C-CH3OLi) which has
been considered to be a factor in lowering specific
activities of the 11C-methylated derivatives. However, in
our hands, in the radiosynthesis of 11C-methionine, the
specific activity was found to be similar using the LAH
method or the 11C-CH4 method. The specific activities
of the various compounds have generally been around
500 Ci/mmol. The 11C-CH4 method is expected to pro-
vide higher specific activities; however it is possible that
contents in the target or the GE box may be affecting
the specific activity of 11C-CO2 which eventually affects
the specific activity of the final product. An approach
Figure 1. Chemical structure of fallypride and fluorine-18 and carbon-
11 radiolabeled isoforms.

J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
97
Figure 2. Reaction scheme for synthesis of the precursor, 5-(3-fluoropropyl)-2-hydroxy-3-methoxy-N-[(2S)-1-(2-propenyl)-2-pyrrolidi-
nyl]methyl]benzamide: (a) MeM chloride, NaH, THF; (b) BH3, THF; (c) 30% H2O2, 1 M NaOH; (d) TsCl, Et3N, CH2Cl2; (e) Bu4N+FÀ, THF; (f)
0.5 N NaOH, THF; (g) diethylaminosulfur trifluoride, CH2Cl2; (h) 8, Et3N, CH2Cl2; (i) TiCl4, CH2Cl2; (j) TFA, CH2Cl2; (k) 8, BOP, Et3N, CH3CN.
currently being employed is to discard the first batch of
11C-CH3I that is produced by the GE box. This possibly
purges the unlabeled CO2 that may be present in the
system and therefore increases the specific activity in the
subsequent runs.
methylation reactions using 11C-methyl iodide. Higher
yields have been reported when 11C-methylations are
carried out with 11C-methyl triflate.11
Chromatographic separation (preparative HPLC, C18
reverse phase column, 56% of acetonitrile, 0.1% of
triethylamine and water at 2.8 mL/min) provided 11C-
fallypride as the principal product (Fig. 4). The HPLC
chromatogram also illustrates the excellent separation
of the phenolic precursor from the radiolabeled pro-
duct, 11C-fallypride which eluted at approximately 10
min. The radiochemical yield of 11C-fallypride was in
the range of 25–40% decay-corrected from 11C-methyl
iodide. Initial specific activities were estimated to range
between 200 and 300 Ci/mmol. Improvements in the
production of 11C-CH3I has now raised the specific
activities of 11C-fallypride to 400–800 Ci/mmol. Thus
far the best specific activity has been 1000 Ci/mmol.
This 11C-CH3I from the GE box was transferred to the
Nuclear Interface 11C-methylation unit (NI box; Fig.
3a) as reported previously.10 Both the GE box and the
NI box were interfaced with a PC controller and were
remotely operated. The 11C-CH3I from the GE box was
transferred to the NI box via a tubing linking the two
boxes and controlled by PC operated valves.
The reaction Scheme used for the radiolabeling proce-
dure is shown in Figure 3b. The reaction vessel in the NI
box contained the phenolic precursor 10 dissolved in
dimethylformamide (DMF) and tetrabutylammonium
hydroxide (TBAH) was used as a base during the radi-
olabeling reaction. This is somewhat similar to the
reaction conditions typically used for the production of
11C-raclopride and 11C-FLB 457 where the base is
sodium hydroxide and reaction solvent is dimethylsulf-
oxide.6 The amount of 11C-CH3I trapped in this reac-
tion vessel typically ranged between 100 and 200 mCi.
The radiolabeling reaction proceeded smoothly with the
desired product, 11C-fallypride as the principal radio-
active species on the HPLC purification (Fig. 4). The
precursor was well separated and the fluorine in the
molecule did not interfere and was not affected by the
radiolabeling conditions. Radiochemical yields were
typically in the 25–40% range and is in the range of 11C-
2.3. PET imaging
Brain uptake and distribution of 11C-fallypride in a
rhesus monkey PET study demonstrated binding in
dopaminergic regions. Example brain slices are shown
in Figure 5. Receptor-rich regions such as putamen were
clearly evident in all the slices. Regions that contain
moderate amount of receptors such as the thalamus
(about 5% of that in the putamen) were also clearly
seen. Cortical regions are known to have small but sig-
nificant amount of D2/D3 receptors as demonstrated in
our previous studies with 18F-fallypride.2 Cortical bind-
ing was also evident with 11C-fallypride.

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J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
Figure 3. (a) GE 11C-methyl iodide module (GE box) and NI 11C-methylation module (NI box) within stacked mini-hot cells. The shielding door is
shown in open position. The 11C-CH3I was produced in the GE box and transferred to the reaction vial in the NI box for the radiolabeling reaction;
(b) radiosynthesis reaction scheme for 11C-fallypride using 11C-CH3I.
Figure 4. HPLC semi-preparative separation profile of fallypride and 11C-fallypride product mixture on a C-18 column (250Â10 mm) eluted with
56% acetonitrle and 44% water containing 0.25% triethylamine at a flow-rate of 2.8 mL/min: (a) UV (254 nm) trace of one fallypride reference
standard (0.016 mg; 43.8 nmol); (b) UV (254 nm) trace of radiolabeling reaction mixture; and (c) radioactivity trace of 11C-fallypride eluted at
approximately 10 min and is well separated from the phenolic precursor appearing at 5 min.

J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
99
Figure 5. Select PET image slices (coronal, transaxial and sagittal planes) of the rhesus monkey brain using 11C-fallypride. Dopaminergic regions
showing 11C-fallypride binding include putamen (Pu), thalamus (T) and cortex (Co).
Decay corrected time–activity curves of 11C-fallypride
in various brain regions of the rhesus monkey brain are
shown in Figure 6 and indicate kinetics that are com-
parable to those observed for 18F-fallypride.2 Highest
uptake is seen in the striatal regions (caudate and
putamen exhibit uptake in the range of 0.04–0.06%
injected dose/cc), with lower uptake in the thalamus
and cortex. Binding in the thalamus and cortex was
found to be somewhat lower than that measured for
18F-fallypride. This is primarily due to the lower spe-
cific activity of 11C-fallypride (200 Ci/mmol for this
particular experiment) compared to that of 18F-fall-
ypride (2800 Ci/mmol). Theroetically however, the
kinetics of the two tracers should be similar if the spe-
cific activity of the 11C-fallypride can be improved.
Similarly, cortical binding was also observed with 11C-
fallypride, but lower than that found for 18F-fallypride.
In the case of caudate and putamen, 11C-fallypride may
not allow enough time to attain equilibrium. Typically
the scanning times of approx. 3 h are employed for
18F-fallypride.1,2
Figure 6. Time–activity curve in rhesus monkey brain after iv admin-
istration of 2.4 mCi 11C-fallypride, specific activity 200 Ci/mmol.
3. Conclusions
1a were prepared according to previously described
methods using 3-methoxysalicylic acid and allyl bro-
mide.7,8 Reactions and extractions solutions were eva-
The synthetic procedure of precursor 5-(3-fluoro-
propyl)-2-hydroxy-3-methoxy-N-[(2S)-1-(2-propenyl)-2-
pyrrolidinyl]methyl]benzamide has been developed. The
compound was found to be stable and suitable for
radiolabeling with 11C-methyl iodide. 11C-Fallypride
was prepared in 25–40% radiochemical yield and spe-
cific activities of 200–1000 Ci/mmol in a tandem GE
11C-CH3I synthesis module (GE box) and NI 11C-
methylation module (NI box). Preliminary 11C-fall-
ypride-PET imaging studies in rhesus monkeys indicate
similar characteristics of uptake and binding to 18F-
fallypride. Our results suggest that 11C-fallypride may
be an excellent PET radiotracer for same day repeat
studies of extrastriatal dopamine D2/D3 receptors.
porated in
a rotatory evaporator under reduced
pressure as appropriate. Analytical and preparative thin
layer chromatography (TLC) were performed using
Baker-flex silica gel IB-F (Philipsburg, NJ, USA) and
Alltech DC-Fertigplatten SIL G-200 UV254 plates
(Deerfield, IL, USA), respectively.
High pressure liquid chromatography (HPLC) was car-
ried out on a Gilson Gradient System (Middleton, WI,
USA) consisting of two Gilson pumps and one UV
detector with wavelength fixed at 254 nm. The system
was used isocratically (varying compositions of acetoni-
trile and water containing triethylamine) for the puri-
fication of radiolabeled reaction mixtures. Semiprep
(250Â10 mm) C18 columns from Alltech were used for
reverse-phase HPLC. Proton nuclear magnetic reso-
nance (NMR, 500 MHz, TMS internal reference in
CDC13) spectra were obtained on a Bruker-Advance
500 spectrometer. Electrospray mass spectra were
obtained on a Model 7250 mass spectrometer (Micro-
mass LCT).
4. Materials and methods
Commercial reagents and solvents (Aldrich Chemical
Company, Milwaukee, WI, USA) of analytical grade
were used without further purification. 2-Hydroxy-3-
methoxy-5-(2-propenyl) benzoic acid, methyl ester 2,
(S)-2(aminomethyl)-N-allylpyrrolidine 8 and fallypride

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J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
1
4.1. 3-Methoxy-2-[(2-methoxyethoxy)methoxy]-5-(2-pro-
penyl)benzoic acid, methyl ester (3)
1:1 hexane: ethyl acetate) as a colorless oil: H NMR d
7.78 (d, 2H, J=8.1 Hz), 7.33 (d, 2H, J=8.3 Hz), 7.19
(d, 1H, J=2.0 Hz), 6.84 (d, 1H, J=2.0 Hz), 5.18 (s,
2H), 4.04 (t, 2H, J=6.0 Hz), 3.95 (s, 3H), 3.87 (s, 3H),
3.83 (s, 2H), 3.57 (m, 2H), 3.37 (s, 3H), 2.63 (m, 2H),
2.45 (s, 3H), 1.96 (m, 2H). Mass spectra (m/z, %): 505
([M+Na]+, 100%), 407 ([M+Na]+–C3H7O2Na, 32%).
2-Hydroxy-3-methoxy-5-(2-propenyl) benzoic acid,
methyl ester 2 (6.00 g; 27 mmol), dissolved in THF (20
mL), was added drop wise to the suspended mixture of
sodium hydride (2.0 g of a 60% (w/w) mineral oil sus-
pension; 139 mmol) in 40 mL of THF at 0 ꢀC and the
reaction was allowed to stir for 30 min at 0 ꢀC. Then (2-
methoxyethoxy)methyl chloride (4 mL, 40.5 mmol) was
added slowly. The reaction was allowed to warm to
room temperature for 2 h. After that the reaction was
quenched by slow addition of 1 M NaOH (40 mL) at
0 ꢀC. The THF was removed by rotary evaporation, and
the product was extracted using CH2Cl2 (3Â50 mL),
dried (MgSO4), filtered, and concentrated. Purification
of the crude material by column chromatography
(gravity silica gel; EtOAc/hexane, 1:l) provided 3 (8 g,
96%; Rf 0.72 in 1:1 hexane/ethyl acetate) as a yellow liquid:
1H NMR d 7.16 (d, 1H, J=2.0 Hz), 6.87 (d, 1H, J=2.0
Hz), 5.93 (ddt, 1H, J=16.7, 10.2, 6.7 Hz), 5.20 (s, 2H),
5.12 (dd, 1H, J=16.7, 1.7 Hz), 5.08 (dd, 1H, J=10.2, 1.6
Hz), 3.96 (m, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.56 (m, 2H),
3.37 (s, 3H), 3.36 (d, 2H). Mass spectra (m/z, %): 333
([M+Na]+, 100%), 235 ([M+Na]+–C3H7O2Na, 15%).
4.4. 3-Methoxy-2-[(2-methoxyethoxy)methoxy]-5-(3-fluoro-
propyl)benzoic acid, methyl ester (6)
Tetra-n-butylammonium fluoride in THF (1 M, 5.1 mL,
5.1 mmol) was added to a solution of tosylate 5 (1.5g,
3.1 mmol) in THF (20 mL). The reaction mixture was
warmed to 55–60 ꢀC and stirred for 2 h. The solution
was concentrated by rotary evaporation and then water
(5 mL) was added and extracted repeatedly with ether
(5Â15 mL). The combined ether extracts were pooled
together, dried (MgSO4) and concentrated, and then
purified by column chromatography (silica gel, EtOAc/
hexane, 1:1) to yield 6 (0.79 g, 77%; Rf 0.67 in 1:1 hex-
ane: ethyl acetate) as a brownish oil. 1H NMR d 7.17 (d,
1H, J=2 Hz), 6.89 (d, 1H, J=2 Hz), 5.2 (s, 2H), 4.50
(dt, 2H, J=47.1, 5.8 Hz), 3.95 (m, 2H), 3.88 (s, 3H),
3.84 (s, 3H), 3.57 (m, 2H), 3.38 (s, 3H), 2.73 (t, 2H,
J=7.5 Hz), 2.01 (m, 2H). Mass spectra (m/z,%): 353
([M+Na]+, 100%).
4.2. 3-Methoxy-2-[(2-methoxyethoxy)methoxy]-5-(3-hy-
droxypropyl)benzoic acid, methyl ester (4)
4.5. 3-Methoxy-2-[(2-methoxyethoxy)methoxy]-5-(3-fluoro-
propyl)benzoic acid (7)
To a cooled (0 ꢀC) solution of alkene 3 (6.00 g; 19.4
mmol) in THF (20 mL) BH3 in THF (1.5 M, 20 mL)
was added, and the mixture was stirred at 0 ꢀC for 30
min followed by 1 h at room temperature. The reaction
mixture was cooled (0 ꢀC) and then 1 M NaOH (50 mL)
was added carefully, followed by 30% aqueous hydro-
gen peroxide (40 mL). The reaction mixture was stirred
at 0 ꢀC for 30 min then room temperature for 1 h. The
THF was removed by rotary evaporation, and the aqu-
eous solution was extracted with CH2C12 (3Â50 mL).
The pooled dichloromethane layers were dried
(MgSO4), filtered, and evaporated to give 5.98g of a
crude oil which was purified by column chromato-
graphy (silica gel; EtOAc/hexane, 1:1) to provide 4 (3.98
g, 63%; Rf 0.29 in 1:1 hexane: ethyl acetate) as a clear
The ester 6 (0.75 g; 3.43 mmol) was dissolved in THF
(20 mL), and then 0.5 M NaOH (60 mL) was added,
and the solution was heated to 80 ꢀC for 1 h. THF was
removed and the aqueous reaction mixture was washed
with ether (20 mL). The aqueous layer was acidified
with H3PO4 to pH 2–3 and then extracted with CH2Cl2
(4Â50 mL). The organic portion was dried (MgSO4)
and filtered, and the solvent was evaporated to give 7
(0.7 g, 100%; Rf 0.45 in 9:1 dichoromethane: methanol)
as a colorless oil. 1H NMR d 7.54 (d, 1H, J=2 Hz), 6.97
(d, 1H, J=2 Hz), 5.2 (s, 2H), 4.47 (dt, 2H, J=47.2, 5.8
Hz), 3.88 (s, 3H), 3.84 (s, 3H), 3.57 (m, 2H), 2.73 (m,
2H), 2.03 (m, 2H). Mass spectra (m/z, %), 339
([M+Na]+, 15%), 242 ([M+H]-C3H7O2]+, 100%)
1
oil. H NMR d 7.17 (d, 1H, J=2.0 Hz), 6.89 (d, 1H,
J=2.0 Hz), 5.2 (s, 2H) 3.96 (m, 2H), 3.87 (s, 3H), 3.84
(s, 3H), 3.68 (t, 2H, J=6.3 Hz), 3.57 (m, 2H), 3.37 (s,
3H), 2.68 (t, 2H, J=7.4 Hz), 1.88 (m, 2H). Mass spectra
(m/z, %): 351 ([M+Na]+, 100%).
4.6. (S)-N-[(1-Allyl-2-pyrolidinyl)methyl]-3-methoxy-2-[(2-
methoxyethoxy)methoxy]-5-(3-fluoropropyl)benzamide (9)
3-Methoxy-2-[(2-methoxyethoxy)methoxy]-5-(3-fluor-
opropyl) benzoyl fluoride (prepared from compound 7
by using reported method),9 (0.35 g, 1.1 mmol) and
Et3N (1.4 mmol, 200 mL) were taken in CH2Cl2 (4 mL).
To this solution (S)-2-(aminomethyl)-N-allylpyrrolidine
(0.15 g, 1.1 mmol) dissolved in CH2Cl2 (1 mL) was
added drop wise at ambient temperature. The reaction
mixture was allowed to stir at ambient temperature for
1 h. The organic layer was washed with saturated solu-
tion of sodium bicarbonate followed by water and was
then dried over MgSO4, filtered and the solvent
removed in vacuo. The crude product was purified by
preparative TLC (CH2Cl2/MeOH, 9:1) to yield 9 (0.24g,
50%; Rf 0.46 in 9:1 dichoromethane/methanol). 1H
4.3. 3-Methoxy-2-[(2-methoxyethoxy)methoxy]-5-(3-[(4-
methylphenyl)sulfonyl]oxy]propyl) benzoic acid, methyl
ester (5)
The alcohol 4 (1.11g; 3.38 mmol) in CH2C12 (5 mL) was
treated with pyridine (0.35 mL, 4.4 mmol) and p-tolu-
enesulfonyl chloride (0.7 g, 3.67 mmol). The reaction vessel
was kept at room temperature for overnight. The reaction
solvent was evaporated, and the residue was treated with
0.25M HCl (20 mL) and extracted with Et2O (2Â25 mL).
The pooled organic layers were dried (MgSO4), filtered,
concentrated, and chromatographed (silica gel column;
EtOAc/hexane, 1:1) to yield 5 (1.54 g, 94%; Rf 0.62 in

J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
101
NMRd 7.46 (d, 1H, J=2 Hz), 6.85 (d, 1H, J=2 Hz),
5.92 (m, 1H), 5.3 (s, 2H), 5.18 (d, 1H), 5.21 (d, 1H), 4.46
(dt, 2H, J=47.1, 5.9 Hz), 3.82 (s, 3H), 3.50 (m, 2H) 3.35
(s, 3H), 3.27 (m, 2H), 3.09 (m, 2H), 2.89 (m, 1H), 2.74
(m, 2H). Mass spectra (m/z, %), 439 ([M+H]+, 100%),
461 ([M+Na]+, 51%).
were pooled together, dried (MgSO4), filtered, evapo-
rated, and purified by preparative TLC (silica gel;
CH2Cl2/MeOH, 9:1) to yield 10 (0.075 g; 48%) as a
yellow mass. Spectral properties confirmed the product
as described before.
4.10. Radiosynthesis
4.7. (S)-N-[(1-Allyl-2-pyrolidinyl)methyl]-2-hydroxy-3-
methoxy-5-(3-fluoropropyl)benzamide (10)
11C-CO2 from a RDS-112 cyclotron was converted to
11C-CH3I in a General Electric methyl iodide unit (GE
box). This 11C-CH3I was then transferred to the reac-
tion vessel in the Nuclear Interface methylation unit (NI
box). The reaction vessel contained a freshly prepared
mixture of the precursor 10 (1 mg, free base) and 1.0 mL
of tetrabutylammonium hydroxide dissolved in 100 mL
DMF and maintained at À10 ꢀC. The reaction was car-
ried out at 50 ꢀC for 2 min and 55 ꢀC for 4 min. The
initial lower temperature of 50 ꢀC (external temperature;
note: bp of CH3I is 41–43 ꢀC) may avoid excessive
vaporization of 11C-CH3I resulting in higher radio-
chemical yield. After the reaction, 0.25 mL of water was
added to the reaction vessel and volatiles were pumped
out. Subsequently, 1 mL of HPLC buffer was added to
the reaction vessel and the contents of the vessel were
loaded and injected onto a C18 column (Alltech Econ-
osil C18 10U, 250Â10 mm) eluted with a solvent con-
taining 56% of acetonitrile, 0.1% of triethylamine and
water at 2.8 mL/min. The radioactive fraction between
10 and 12 min, which corresponds to the reference fall-
ypride under the same condition, was collected. This
collected fraction was taken to near dryness in vacuo.
Approximately 5–8 mL of sterile saline (0.9%, NaCl
INJ, USP, 10 mL single-dose, Abbot Laboratories,
Chicago, IL, USA) was added to the flask. The contents
of the flask were then drawn into a 5- or 10-mL sterile
syringe depending on volume. The contents of the syr-
inge were then filtered through a 0.2 micron Millex-FG
Millipore sterile filter (Millipore Corp., Bedford, MA,
USA). This final dose is then used to determine specific
activity using the fallypride standards.
Benzamide 9 (0.19 g; 0.46 mmol) was dissolved in
CH2Cl2 (5 mL) and TiC14 in CH2Cl2 (1 M, 2.5mL) was
added at 0 ꢀC. The mixture was stirred for 1 h at 0 ꢀC. A
10% solution of NaHCO3 (25 mL) was added slowly at
0 ꢀC. The solution was then warmed to room tempera-
ture. The reaction mixture was extracted with CH2Cl2
(3Â25 mL). The organic portions were pooled together,
dried (MgSO4), filtered, evaporated, and purified by
preparative TLC (silica gel; CH2Cl2:MeOH, 9:1) to
yield 10 (0.11 g, 76%; Rf 0.50 in 9:1 dichoromethane:
methanol) as a yellow mass. 1H NMR d 8.05 (br, 1H), 7.09
(s, 1H), 6.83 (d, 1H, J=1.7 Hz), 5.98 (ddt, 3H, J=16.8,
10.1, 6.8 Hz), 4.47 (dt, 2H, J=47.3, 5.9 Hz), 3.88 (s, 3H),
3.52 (t, 2H, J=6.3 Hz) 3.35 (s, 3H), 3.27 (m, 2H), 3.09 (m,
2H), 2.89 (m, 1H), 2.74 (m, 2H). Mass spectra (m/z, %),
351 ([M+H]+, 100%), 373 ([M+Na]+, 3%).
Closer examination of this product revealed a mixture
of the chloro- and fluoro-derivatives, which were insep-
arable on preparative TLC. An alternate method of
deprotection of the MEM group involved the use of
trifluoroacetic acid that is described below.
4.8. 2-Hydroxy- 3-methoxy -5-(3-fluoropropyl)benzoic
acid (11)
The acid 7 (0.2 g; 0.88 mmol) was dissolved in dichor-
omethane (1 mL) and 0.2 mL of trifluoroacetic acid was
added. This mixture was allowed to stir overnight after
which time the contents were dried in vacuo. The resi-
due was taken up in 10% NaHCO3 and acidified with 1
N HCl and extracted with dichoromethane. The organic
portions were pooled together, dried (MgSO4), filtered,
evaporated, and purified by preparative TLC (silica gel;
CH2Cl2:MeOH, 9:1) to yield 11 (0.10 g, 50%; Rf 0.49 in
9:1 dichoromethane: methanol) as a yellow oil. 1H
NMR d 7.59 (d, 1H, J=1.9 Hz), 6.98 (d, 1H, J=2 Hz),
4.48 (dt, 2H, J=47.1, 5.9 Hz), 3.92 (s, 3H), 2.74 (m,
2H), 2.06 (m, 2H). Mass spectra (m/z, %), 229
([M+H]+, 3%), 251 ([M+Na]+, 100%).
4.11. PET imaging
The rhesus monkey was anesthetized using ketamine (10
mg/kg), xylazine (0.5 mg/kg) and was subsequently
maintained on 0.5–1.5% isoflurane. Two intravenous
catheters were placed, one on each arm, for purposes of
administration of the radiopharmaceutical and for
obtaining blood samples during the study. The head of
the animal was placed in the gantry of a ECAT EXACT
HR+ PET scanner and positioned in place with the use
of adhesive tape as described previously.2 A transmis-
sion scan using a Ge-68/Ga-68 rod source was acquired
prior to administration of the radiopharmaceutical to
correct for tissue attenuation of the coincident radia-
tion. A dynamic sequence of scans for a total of
approximately 70 min was acquired in the 3-D-mode
immediately after administering approximately 2–3 mCi
of 11C-fallypride intravenously. Data in final form were
expressed in units of percent-injected dose/cc or nCi/cc.
Areas showing maximal radioligand binding within the
caudate, putamen, thalamus, cortex and cerebellum
were delineated in the images.
4.9. (S)-N-[(1-allyl-2-pyrolidinyl)methyl]-2-hydroxy-3-
methoxy-5-(3-fluoropropyl)benzamide (10)
The acid 11 (0.1 g; 0.44 mmol) was dissolved in aceto-
nitrile (1 mL) and to this was added (S)-2-(aminoethyl)-
N-allylpyrrolidine (0.07g, 0.55 mmol). While stirring,
BOP reagent (benzotriazol-1-yloxy-tris(dimethylamino)-
phosphonium hexafluorophosphate) 0.195 g and 0.1 mL
of triethylamine were added and the reaction mixture
was stirred overnight. Solvents were removed in vacuo
and the residue was taken up in dichloromethane and
washed with water and NaHCO3. The organic portions

102
J. Mukherjee et al. / Bioorg. Med. Chem. 12 (2004) 95–102
3. Mukherjee, J.; Yang, Z. Y.; Lew, R.; Brown, T.; Kronmal,
Acknowledgements
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4. Christian, B.; Narayanan, T.; Shi, B.; Metz, J.; Mukherjee,
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J. Nucl. Med. 1995, 36, 1275.
This research was supported by the Biological and
Environmental Program (BER), US Department of
Energy, Grant No. DE-FG03-02ER63294. We would
like to acknowledge the Wallace-Kettering Neuroscience
Institute and the Air Force Research Laboratory under
Cooperative Agreement No. F33615-98-2-6002 for use
of the PET scanner. We like to thank Steve Mattmuller
for technical assistance with the GE and NI boxes.
7. Bishop, J. E.; Mathis, C. A.; Gerdes, J. M.; Whitney,
J. M.; Eaton, A. M.; Mailman, R. B. J. Med. Chem. 1991,
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References and notes
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