Synthesis of 1-(2,4-dichlorophenyl)-4-cyano-5-(4-[11C] methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide ([ 11C]JHU75528) and 1-(2-bromophenyl)-4-cyano-5-(4-[ 11C]methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-c

Article abstract of DOI:10.1002/jlcr.1125

Two novel ligands for cerebral cannabinoid receptor (CB1), 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl) -1H-pyrazole-3-carboxamide (JHU75528) and 1-(2-bromophenyl)-4-cyano-5-(4- methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carb

Full text of DOI:10.1002/jlcr.1125

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 1021–1036.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1125
Research Article
Synthesis of 1-(2,4-dichlorophenyl)-4-cyano-5-(4-
[11C]methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-
3-carboxamide ([11C]JHU75528) and 1-(2-
bromophenyl)-4-cyano-5-(4-[11C]methoxyphenyl)-
N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide
([11C]JHU75575) as potential radioligands for
PET imaging of cerebral cannabinoid receptor
Hong Fan*, Hayden T. Ravert, Daniel P. Holt, Robert F. Dannals and
Andrew G. Horti
Department of Radiology, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA
Summary
Two novel ligands for cerebral cannabinoid receptor (CB1), 1-(2,4-dichlorophenyl)-4-
cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (JHU75528)
and 1-(2-bromophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-
carboxamide (JHU75575) have been synthesized. Both JHU75528 and JHU75575
display a combination of higher binding affinity and lower lipophilicity than those of
Rimonabant (SR141716), a high affinity CB1 selective antagonist, and AM281, the only
available ligand for emission tomography imaging of CB1 in human subjects.
Radiolabeled [11C]JHU75528 and [11C]JHU75575 were prepared by reaction of
[11C]methyl iodide with nor-methyl precursors. The average radiochemical yield, specific
radioactivity, and radiochemical purity of [11C]JHU75528 were 16%, 235 GBq/mmol
(6360 mCi/mmol), and 99%, respectively; those of [11C]JHU75575 were 8%, 196 GBq/
mmol (5308mCi/mmol), and 99%, respectively. Both ligands hold promise as PET
radioligands for imaging CB1 receptor. Copyright # 2006 John Wiley & Sons, Ltd.
Received 11 July 2006; Revised 2 August 2006; Accepted 2 August 2006
Key Words: cannabinoid receptor; carbon-11; positron emission tomography;
Rimonabant; SR141716
*Correspondence to: H. Fan, Department of Radiology/PET Center, 720 Rutland Ave. Ross Bldg. 320,
Baltimore, MD 21205, USA. E-mail: hfan@jhmi.edu
Contract/grant sponsor: Department of Radiology
Copyright # 2006 John Wiley & Sons, Ltd.

1022
H. FAN ET AL.
1. Introduction
The development of the radiotracers for emission tomography imaging of
cerebral cannabinoid receptor (CB1) is important for studying the role of CB1
in schizophrenia,1–4 obesity,5–8 drug dependence9,10 and a variety of other
disorders11 as well as for developing cannabinergic medications to treat these
conditions.12–16 The radiotracers studied to date, mainly analogs of
Rimonabant and WIN 55,212-2, have exhibited properties that were
inadequate for successful quantitative tomography imaging due to low specific
binding, high non-specific binding, and insufficient brain uptake of the
radiotracers.17–29
Low binding potentials of all existing CB1 radioligands are due to either
insufficient binding affinity or too high lipophilicity (log D>4) of these
compounds.18,20,25 Even though many other properties of CNS radioligands
are crucially important,30 it is essential to develop a CB1 radioligand with a
combination of high binding affinity and reduced lipophilicity (log D54). It
was our hypothesis that the structure of one of the best currently available
CB1 PET radioligand 1-(2,4-dichlorophenyl)-5-(4-[11C]methoxyphenyl)-
4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide ([11C]NIDA41020 or
[11C]SR149080) (Figure 1),19,20,31 a methoxy-analog of Rimonabant, could be
modified in such a way that the new ligand would exhibit high binding affinity
and lower lipophilicity. We attempted to identify a position in the Rimonabant
molecule that would tolerate hydrophilic substituents.20 In our earlier studies we
found that introduction of hydrophilic substituents in the 5-phenyl ring of
Rimonabant caused a reduction of the binding affinity.20 Recent studies by
other groups demonstrated that non-pyrazole analogs of Rimonabant are
tolerant to the attachment of CN-group in the central core ring.32–34 Because a
cyano-group is hydrophilic,35 we chose to introduce this group in the pyrazole
core of NIDA41020 and determine the binding affinities and lipophilicities of
the cyano-analogs. We targeted a compound with higher binding affinities and
lower lipophilicities than those of AM281 (Figure 1), the only available ligand
for emission tomography imaging of CB1 in human subjects.27 Here we present
development of 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piper-
idin-1-yl)-1H-pyrazole-3-carboxamide (JHU75528) and its monobromo-analog
(JHU75575) (Figure 2), two CB1 radioligands with high binding affinities and
reduced lipophilicities as compared with AM281.
Results and discussion
Chemistry
JHU75528, 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-
1-yl)-1H-pyrazole-3-carboxamide 1a, and JHU75575, 1-(2-bromophenyl)-4-
cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide 1b,
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1023
Figure 1. Representative cannabinoid ligands and radioligands
Figure 2. Chemical structures
were synthesized via
a
conventional method of preparation of
4-cyanopyrazole derivatives36–39 as shown in Scheme 1. Briefly, 4-methox-
ybenzoylacetonitrile underwent cycloaddition reaction with chloro[(2,4-
dichlorophenyl)hydrazono]ethyl acetate39 or chloro[(2-bromophenyl)hydrazo-
no]ethyl acetate 7 in the presence of sodium ethoxide to give pyrazole
derivatives 4a or 4b. The cycloaddition products 4a and 4b were saponified with
lithium hydroxide to give the carboxylic acids 5a and 5b, which were converted
to the carbonyl chloride intermediates followed by coupling with 1-
aminopiperidine to afford 1a or 1b in reasonable yields. Chloro[(2-
bromophenyl)hydrazono]ethyl acetate 7 was prepared in two steps (Scheme
2). 2-Bromo aniline was first treated with 24% HCl and NaNO2 to yield
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1024
H. FAN ET AL.
Scheme 1. Synthesis of precursors and target compounds
Scheme 2. Synthesis of Chloro[(2-bromophenyl)hydrazono]ethyl-acetate
arenediazonium salt. Then the diazonium salt was converted to chloro[(2-
bromophenyl)hydrazono]ethyl acetate by reacting with ethyl 2-chloro-acetoa-
cetate.39
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1025
Attempts to generate nor-methyl precursors 6a (JHU75555) and 6b
(JHU75580) for radiosynthesis either via demethylation or by a direct
synthesis were not successful. Therefore, preparation of precursors required
a protective group approach. Among the several protective groups including
t-butyl carbamate (Boc), triisopropylsilyl (TIPS), and t-butyl, we found that
allyl group was the best protective group for this reaction scheme. The
intermediate for this synthesis, 3-(4-(allyloxy)phenyl)-3-oxopropanenitrile 3,
was obtained by treating ethyl 4-hydroxybenzoate with allyl bromide, and
then condensing with acetonitrile in the presence of sodium ethoxide. In the
final step, 6a (JHU75555) and 6b (JHU75580) were prepared via Pd-catalyzed
removal of allylic protective group of 1c and 1d in the presence of
phenyltrihydrosilane.40
Binding affinity and lipophilicity
In the inhibition binding assay experiments, JHU75528 and JHU75575
displayed substantially higher binding affinities than those of AM281 and
Rimonabant (Table 1).
The conventional criterion for selection of CNS receptor radiotracer, the
value of Bmax/Ki, has to be 10 or greater.41 Assuming that the Bmax of CB1
receptors in the mammalian brain reaches values above 1 pmol/mg tissue
(10ꢀ6 M),14 the Bmax/Ki ratios of JHU75528 and JHU75575 are fairly high
(see Table 1).
Table 1. Inhibition binding affinity ([3H]CP55940, recombinant hCB1) and lipophilicity
of Rimonabant (SR141716), AM281 and JHU75528
Compound Binding affinity, Bmax/Kia Calculated Experimental
Target-to-non-
target ratio/time
b
Ki, (nM)
log D7.4
log D7.4
Rimonabant 35.4
46.2
}
6.0
4.6 ꢁ 0.8(4)c
}
AM281
422
525
2.4
1.9
91
4.9
4.3
3.8
}
2.0 (mice)/2 h[26]f
0.22 (human)/[27]g
JHU75528
11 ꢁ 7 (3)d
3.6 ꢁ 0.3 (4)c 3.4 (mice)/2 h[42]h
3.3 ꢁ 0.02 (4)e 2.5(baboon)/1.5 h[42]i
}
JHU75575
4.7
213
3.4 ꢁ 0.1(8)e
a Bmax=10ꢀ6 M14 and Ki unit is M.
b Calculated by ACD/logD software (Advanced Chemistry Development Inc., Toronto).
c The octanol/phosphate buffer (pH=7.4) partition coefficient of non-labeled JHU75528 was measured in
four assays (mean ꢁ S.D.) using conventional flask-shake technique.
d Mean ꢁ S.D. (number of independent assays).
e The partition coefficient between octanol and phosphate buffer (pH=7.4) was measured using
radioactivity-counting procedure43
f Cerebellum/brainstem.
.
g Estimated value of the binding potential (BP) in the lentiform nuclei with white matter as the reference
region.
h Striatum/brainstem.
i Putamen/pons.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1026
H. FAN ET AL.
Lipophilicities of JHU75528, JHU75575, AM281, and Rimonabant were
calculated and determined experimentally (Table 1). Unlike the previously
studied PET radioligands for imaging CB1, the lipophilicities of JHU75528
and JHU75575 are lower than those of AM281 and Rimonabant and they fall
in the range that is conventionally optimal for BBB permeability.
These in vitro studies demonstrate that both JHU75528 and JHU75575 are
CB1 ligands with a combination of high binding affinity and relatively low
lipophilicity. This combination of the ligand properties and the high density of
CB1 receptors in mammalian brain14 suggest that radiolabeled [11C]JHU75528
and [11C]JHU75575 could label CB1 receptors in vivo with high specific
binding and low non-specific binding. Imaging properties of [11C]JHU75528
have been recently evaluated in rodents and baboon.42 This radioligand holds
promise as the first PET radiotracer for quantitative PET imaging of CB1
receptor in human subjects.
Radiochemistry
Radiomethylation of 6 with [11C]iodomethane at 808C in acetone in the
presence of 2 M NaOH proved straightforward (Scheme 3). The average non-
decay corrected radiochemical yields based on trapped radio-activities of
[11C]CH3I for [11C]1a and [11C]1b were 16 ꢁ 5% (n ¼ 12) and 8.2 ꢁ 1%
(n ¼ 4), respectively, which are comparable to C-11 methylations reported on
other members of the phenolic series.44, 45 The non-optimized time of synthesis
for [11C]1a and [11C]1b, including HPLC purification and formulation as a
sterile solution in saline containing 10% ethanol was approximately 28 min.
Radiochemical purity and specific activity were determined by analytical
HPLC. Specific activity was calculated by relating the area of the UV
absorbance peak of carrier ligand in an aliquot of known radioactivity to the
area of a standard sample of ligand. The final radioligands were obtained with
radiochemical purity greater than 99% and specific activities 235 ꢁ 108 GBq/
Scheme 3. Radio-synthesis
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1027
mmol (6360 ꢁ 2914 mCi/mmol) for [11C]1a in 11 production runs, and
196 ꢁ 15 GBq/mmol (5308 ꢁ 419 mCi/mmol) for [11C]1b in 4 runs at the end-
of-synthesis.
Materials and methods
General
DMF was prepared by sequential distillation under reduced pressure from
CaH2 and BaO. All other chemicals and solvents were reagent grade, and were
1
used as received from Aldrich. H NMR spectra were obtained with a Vivian
400 MHz spectrometer. Chemical shifts are reported in ppm (d) relative to
internal tetramethylsilane in CDCl3. High resolution mass spectrometry was
performed at the University of Notre Dame Mass Spectrometry Facility.
Elemental analysis was done by Galbraith Laboratories Inc. (Knoxville, TN).
Flash chromatography purification was performed using E. Merck 7729
(5230 mesh) silica gel. [11C]iodomethane was prepared with the PETtrace MeI
MicroLab (GE, Milwaukee, WI) using a PETtrace cyclotron. HPLC analyses
and purification for [11C]JHU75528 and [11C]JHU75575 were performed with
two model 590EF HPLC pumps (Waters, Milford, MA), an in-line model 441
fixed wavelength (254 nm, Waters) ultraviolet (UV) detector, and a flow-
through 2-in NaI(Tl) crystal scintillation detector (model 276; Ortec, Oak
Ridge, TN). All HPLC chromatograms were recorded by a Varian Galaxie
system. Semi-preparative (10 mm ꢂ 250 mm) and analytical (4.6 mm ꢂ
100 mm) 10 mm C-18 Luna columns (Phenomenex Torrance, CA) were used
for purification and quality control, respectively.
Chloro[(2-bromophenyl)hydrazono]ethyl acetate (7)
A mixture of 2-bromo-aniline (7.8 g, 45 mmol) in 75 ml 24% HCl, and 200 ml
water was stirred for 2 h at room temperature. A solution of sodium nitrite
(3.17 g, 46 mmol) in 21 ml water was added dropwise to the reaction mixture,
which was cooled with ice, for 30 min. The resulting solution was treated with
a solution of sodium acetate (3.51 g, 43 mmol) and ethyl 2-chloro-acetoacetate
(6.21 ml, 45 mmol) in 450 ml EtOH, then cooled with ice. The temperature was
allowed to slowly increase for 2 h. The precipitate was filtered, washed with
1
water and dried to give desired product 12.5 g, (91%). H NMR (CDCl3, d)
1.41 (t, J = 6.8 Hz, 3H), 4.40 (q, J = 6.8 Hz, 2H), 6.91 (m, 1H), 7.33 (m, 1H),
7.51 (dd, J1 = 8.0 Hz, J2 = 1.6 Hz, 1H), 7.62 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz,
1H), 8.86 (b, 1H, NH). FAB-HRMS: calculated for C10H10BrClN2O2:
303.9614; found: 303.9638.
Ethyl 4-(allyloxy)benzoate (2)
Ethyl 4-hydroxybenzoate (2.32 g, 14 mmol), allyl bromide (1.69 ml, 20 mmol),
and potassium carbonate (2.84 g, 20 mmol) in acetone (50 ml) were heated at
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1028
H. FAN ET AL.
reflux for 3 h. The reaction solution was cooled and filtered. The filtrate was
evaporated to dryness to yield colorless oil 2.90 g, 100%. 1H NMR (CDCl3, d)
1.37 (t, J = 7.2 Hz, 3H), 4.34 (dd, J1 = 8.4 Hz, J2 = 7.2 Hz, 2H), 4.58(m, 2H),
5.31 (m, 1H), 5.42 (m, 1H), 6.04 (m, 1H), 6.92 (dd, J1 = 7.2 Hz, J2 = 1.6 Hz,
2H), 7.99 (dd, J1 = 6.8 Hz, J2 = 1.6 Hz, 2H). FAB-HRMS: calculated for
C12H14O3: 206.2378; found: 207.1022 (M+H)+.
3-(4-(allyloxy)phenyl)-3-oxopropanenitrile (3)
A mixture of ethyl (4-allyloxy) benzoate 2 (2.06 g, 10 mmol), sodium ethoxide
(0.748 g, 11 mmol), and acetonitrile (0.65 ml, 12 mmol) in anhydrous toluene
(5 ml) was stirred at 1108C for 20 h. The reaction was cooled and diluted with
30 ml water to dissolve solid. The aqueous layer was washed with Et2O
(2 ꢂ 30 ml), then was acidified with 1 N HCl to pH 7. The solution was
extracted with CH2Cl2 (3 ꢂ 30 ml). The organic phase was washed with brine
and dried over Na2SO4. The solvent was then removed to afford the desired
1
product 555 mg, 27.6%. H NMR (CDCl3, d) 4.02 (s, 2H), 4.63(m, 2H), 5.35
(m, 1H), 5.46 (m, 1H), 6.05 (m, 1H), 6.99 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 2H),
7.99 (dd, J1 = 7.2 Hz, J2 = 2.0 Hz, 2H). FAB-HRMS: calculated for
C12H11NO2: 201.2212; found: 202.0854(M+H)+.
Ethyl 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-car-
boxylate (4a)
A mixture of chloro[(2,4-dichlorophenyl)hydrazono]ethyl acetate39 (1.63 g,
5.5 mmol), 4-methoxybezoylacetonitrile (0.97 g, 5.5 mol) , 60 ml EtOH, and
sodium ethoxide, which was prepared by dissolving 0.14 g sodium in 12.5 ml
EtOH, was heated to reflux for 18 h. After cooling to room temperature, the
solvent was removed by reduced pressure. EtOAc (75 ml) was added and the
precipitate was filtered. The organic solution was washed with water and
brine, dried over Na2SO4, and evaporated with a rotary evaporator. The crude
product was purified by flash chromatography 10:90 EtOAc/ CH2Cl2 to afford
1
the desired product 440 mg, 19%. H NMR (CDCl3, d) 1.46 (t, J = 6.0 Hz,
3H), 3.81 (s, 3H), 4.52 (dd, J1 = 14.4 Hz, J2 = 2.8 Hz, 2H ), 6.89 (dd, J1 =
6.8 Hz, J2 = 2.0 Hz, 2H), 7.26 (dd, J1 = 7.2 Hz, J2 = 2.4 Hz, 2H), 7.37 (m,
1H), 7.42 (s,1H), 7.46 (m, 1H). FAB-HRMS: calculated for C20H15Cl2N3O3:
415.0490; found: 416.0569 (M+H)+.
Ethyl 1-(2-bromophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carbo-
xylate (4b)
Compound 4b was prepared from chloro[(2-bromophenyl)hydrazono]ethyl
acetate 7 with the same procedure described for 4a, to afford the desired
product 15%. 1H NMR (CDCl3, d) 1.47 (t, J = 7.2 Hz, 3H), 3.80 (s, 3H), 4.52
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1029
(q, J = 1.2 Hz, 2H), 6.86 (d, J = 8.8 Hz), 7.28 (d, J = 8.8 Hz), 7.36 (m, 1H),
7.44 (m, 2H), 7.62 (m, 1H). FAB-HRMS: calculated for C20H16BrN3O3:
425.0375; found: 425.0359.
Ethyl 5-(4-(allyloxy)phenyl)-1-(2,4-dichlorophenyl)-4-cyano-1H-pyrazole-3-
carboxylate (4c)
Compound 4c was prepared from 3-(4-(allyloxy)phenyl)-3-oxopropanenitrile 3
and chloro[(2,4-dichlorophenyl)hydrazono]ethyl acetate39 with the same
1
procedure described for 4a, to afford the desired product 34%. H NMR
(CDCl3, d) 1.46 (t, J = 6.8 Hz, 3H), 4.53(m, 4H), 5.31 (m, 1H), 5.40 (m, 1H),
6.00 (m, 1H), 6.90 (dd, J1 = 6.8 Hz, J2 = 2.0 Hz, 2H), 7.25 (dd, J1 = 7.2 Hz,
J2 = 2.4 Hz, 2H), 7.37 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 7.42 (d, J = 8.4 Hz
1H), 7.46 (d, J = 2.4 Hz, 1H). FAB-HRMS: calculated for C22H17Cl2N3O3:
441.0647; found: 442.0742 (M+H)+.
Ethyl 5-(4-(allyloxy)phenyl)-1-(2-bromophenyl)-4-cyano-1H-pyrazole-3-car-
boxylate (4d)
Compound 4d was prepared from 3-(4-(allyloxy)phenyl)-3-oxopropanenitrile 3
and chloro[(2-bromophenyl)hydrazono]ethyl acetate 7 with the same proce-
dure described for 4a, to afford the desired product 21%. 1H NMR (CDCl3, d)
1.47 (t, J = 6.8 Hz, 3H), 4.53(m, 4H), 5.30 (m, 1H), 5.39 (m, 1H), 6.01 (m,
1H), 6.86 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 8.8 Hz, 2H), 7.34 (m, 1H), 7.42 (m,
2H), 7.46 (dd, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H). FAB-HRMS: calculated for
C22H18BrN3O3: 451.0532; found: 451.0514.
1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic
acid (5a)
A mixture of ethyl 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-
pyrazole-3-carboxylate 4a (0.51 g, 1.22 mmol) in 35 ml THF and LiOH (35 mg,
1.5 mmol) in 3 ml water was heated to 658C for 2 h. After cooling to room
temperature, 25 ml of cold water and 2.5 ml of HCl (5%) were added. The
solution was extracted with dichloromethane (3 ꢂ 50 ml). The organic layer
was washed with brine and dried over Na2SO4. The solvent was removed to
1
afford the desired product 432 mg, 91%. H NMR (CDCl3, d) 3.82 (s, 3H),
6.90 (dd, J1 = 7.2 Hz, J2=2.0 Hz, 2H), 7.26 (dd, J1 = 7.2 Hz, J2 = 2.4 Hz,
2H), 7.39 (d, J = 2.0 Hz, 1H), 7.41 (s, 1H), 7.48 (d, J = 2.0 Hz, 1H). FAB-
HRMS: calculated for C19H11Cl2N3O3: 387.0177; found: 388.0238 (M+H)+.
1-(2-bromophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic
acid (5b)
Compound 5b was prepared from 4b with the same procedure described for
5a, to afford the desired product 96%. 1H NMR (CD3OD, d) 3.79 (s, 3H), 6.93
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1030
H. FAN ET AL.
(d, J = 8.8 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 7.49 (m, 2H), 7.63 (m, 1H), 7.71
(m, 1H). FAB-HRMS: calculated for C18H12BrN3O3: 397.0062; found:
397.0063.
5-(4-(allyloxy)phenyl)-1-(2,4-dichlorophenyl)-4-cyano-1H-pyrazole-3-carbo-
xylic acid (5c)
Compound 5c was prepared from 4c with the same procedure described for 5a,
1
to afford the desired product 97%. H NMR (CDCl3, d) 4.54 (d, J = 4.8 Hz,
2H), 5.31 (m, 1H), 5.41 (m, 1H), 6.03 (m, 1H), 6.90 (dd, J1 = 6.8 Hz, J2 =
2.0 Hz, 2H), 7.24 (dd, J1 = 7.2 Hz, J2 = 2.4 Hz, 2H), 7.40 (m, 2H), 7.48 (d, J
= 2.4 Hz, 1H). FAB-HRMS: calculated for C20H13Cl2N3O3: 413.0334; found:
414.0391 (M+H)+.
5-(4-(allyloxy)phenyl)-1-(2-bromophenyl)-4-cyano-1H-pyrazole-3-carboxylic
acid (5d)
Compound 5d was prepared from 4d with the same procedure described for
1
5a, to afford the desired product 66%. H NMR (CDCl3, d) 4.51 (d, J =
4.8 Hz, 2H), 5.30 (dd, J1 = 10.8 Hz, J2 = 1.6 Hz, 1H), 5.39 (dd, J1 = 17.2 Hz,
J2 = 1.6 Hz, 1H), 6.03 (m, 1H), 6.86 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz,
2H), 7.36 (m, 1H), 7.44 (m, 2H), 7.61 (d, J = 8.0 Hz, 1H). FAB-HRMS:
calculated for C20H14BrN3O3: 423.0219; found: 423.0212.
1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-
pyrazole-3- Carboxamide (1a)
A mixture of a solution of 1-(2,4-dichlorophenyl)-4-cyano-5-(4-methoxyphe-
nyl)-1H-pyrazole-3-carboxylic acid 5a (0.19 g, 0.5 mmol) in toluene (5 ml) and
thionyl chloride (0.1 ml, 1.33 mol) was refluxed for 2 h. The solvent was
removed under reduced pressure. Extra toluene (5 ml) was added and
evaporated under vacuum. The crude acid chloride in CH2Cl2 (5 mL) was
cooled to 08C, and Et3N (0.1 ml, 0.71 mmol) with 1-amino piperidine (79 mL,
0.71 mmol) added. The solution was stirred at room temperature for 20 h. The
reaction mixture was diluted with CH2Cl2, and washed the organic phase
washed with brine, dried over Na2SO4. The solvent was removed and the crude
product was purified by flash chromatography 1:3 EtOAc/hexanes to afford
1
the desire product 87 mg, 36.7%. H NMR (CDCl3, d) 1.43 (b, 2H), 1.74 (m,
4H), 2.89 (b, 4H), 3.81 (s, 3H), 6.87 (dd, J1=6.8 Hz, J2=2.4 Hz, 2H), 7.23 (dd,
J1=6.8 Hz, J2=2.4 Hz, 2H), 7.41 (d, J=2.0 Hz, 1H), 7.43 (d, J =7.6 Hz, 1H),
7.48 (d, J =2.0 Hz, 1H), 7.57 (b, 1H). FAB-HRMS: calculated for
C23H21N5O2Cl2: 469.1072; found: 470.1125 (M+H)+. Analytical
*
(C23H21N5O2Cl2 0.55EtOAc) C, H, N.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1031
1-(2-Bromophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyra-
zole-3-carboxamide (1b)
Compound 1b was prepared from 5b with the same procedure described for
1a, to afford the desired product 26%. 1H NMR (CDCl3, d) 1.43 (b, 2H), 1.75
(m, 4H), 2.85 (b, 4H), 3.79 (s, 3H, CH3), 6.84 (d, J = 8.4 Hz), 7.25 (d,
J=8.8 Hz), 7.43 (m, 3H), 7.60 (b, 1H), 7.66 (d, J = 7.6 Hz, 1H). FAB-HRMS:
calculated for C23H22BrN5O2: 479.0597; found: 480.1028 (M+H)+. Analy-
ꢃ
tical (C23H22BrN5O2 0.4EtOAc ꢃ 0.3CH2Cl2) C, H, N.
5-(4-(allyloxy)phenyl)-1-(2,4-dichlorophenyl)-4-cyano-N-(piperidin-1-yl)-1H-
pyrazole-3-carboxamide (1c)
Compound 1c was prepared from 5c with the same procedure described for 1a,
to afford the desired product 41%. 1H NMR (CDCl3, d) 1.44 (b, 2H), 1.75 (m,
4H), 2.91 (b, 4H), 4.53 (d, J = 4.6 Hz, 2H), 5.31 (d, J = 9.2 Hz, 1H), 5.40 (d, J
= 18.8, Hz, 1H), 6.02 (m, 1H), 6.88 (dd, J1 = 11.2 Hz, J2 = 2.4 Hz, 2H), 7.22
(dd, J1 = 7.2 Hz, J2 = 1.6 Hz, 2H), 7.38 (m, 2H), 7.49 (d, J = 2.0 Hz, 1H),
7.55 (b, 1H). FAB-HRMS: calculated for C25H23Cl2N5O2: 495.1229; found:
496.1294 (M+H)+.
5-(4-(allyloxy)phenyl)-1-(2-bromophenyl)-4-cyano-N-(piperidin-1-yl)-1H-
pyrazole-3-carboxamide (1d)
Compound 1d was prepared from 5d with the same procedure described for
1a, to afford the desired product 50%. 1H NMR (CDCl3, d) 1.44 (b, 2H), 1.76
(m, 4H), 2.90 (b, 4H), 4.51 (m, 2H), 5.30 (dd, J1 = 10.4 Hz, J2 = 1.2 Hz, 1H),
5.39 (dd, J1 = 10.4 Hz, J2 = 1.2 Hz, 1H), 6.01 (m, 1H), 6.86 (m, 2H), 7.26 (m,
2H), 7.39 (m, 3H), 7.59 (s, 1H), 7.67 (d, = 7.6 Hz, 1H). FAB-HRMS:
calculated for C25H23BrN5O2: 491.1004; found: 491.1082.
1-(2,4-dichlorophenyl)-4-cyano-5-(4-hydroxyphenyl)-N-(piperidin-1-yl)-1H-
pyrazole-3-carboxamide (6a)[40]
A mixture of 5-(4-(allyloxy)phenyl)-1-(2,4-dichlorophenyl)-4-cyano-N-(piper-
idin-1-yl)-1H-pyrazole-3-carboxamide 1c (324 mg, 0.65 mmol), Pd(PPh3)4
(15 mg, 0.013 mmol), and PhSiH3( 140 mg, 1.3 mmol) in 20 ml CH2Cl2 was
stirred at room temperature for 1 h. The reaction was monitored by TLC (1:1
EtOAc/hexanes). After the reaction was complete, the solvent was removed
under reduced pressure. EtOAc (50 ml) was added to dissolve the residue. The
organic layer was washed with saturated NaHCO3, brine, dried over Na2SO4
and evaporated to an oil. The residue was dissolved and purified by flash
1
chromatograph 1:1 EtOAc/CH2Cl2, to yield 287 mg, 96%. H NMR (CDCl3,
d) 1.42 (b, 2H), 1.73 (m, 4H), 2.87 (b, 4H), 6.87 (dd, J1 = 6.4 Hz, J2 = 1.6 Hz,
2H), 7.16 (dd, J1 = 6.4 Hz, J2 = 1.6 Hz, 2H), 7.37 (m, 2H), 7.49 (d, J =
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1032
H. FAN ET AL.
2.0 Hz, 1H), 7.59 (b, 1H). FAB-HRMS: calculated for C22H19Cl2N5O2:
455.0916; found: 456.1013 (M+H)+.
1-(2-bromophenyl)-4-cyano-5-(4-hydroxyphenyl)-N-(piperidin-1-yl)-1H-pyra-
zole-3-carboxamide (6b)
Compound 6b was prepared from 1d with the same procedure described for
6a, to afford the desired product 75%. 1H NMR (CDCl3, d) 1.37 (b, 2H), 1.66
(m, 4H), 2.82 (b, 4H), 6.89 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 7.38
(m, 3H), 7.64 (d, J = 7.6 Hz, 1H), 7.68 (s, 1H), 8.23 (b, 1H). FAB-HRMS:
calculated for C22H20BrN5O2: 465.0800; found: 466.0855 (M+H)+.
1-(2,4-dichlorophenyl)-4-cyano-[11C]5-(4-methoxyphenyl)-N-(piperidin-1-yl)-
1H-pyrazole-3- carboxamide (½11Cꢄ1a)
To a 1 ml V-vial was added 1-(2,4-dichlorophenyl)-4-cyano-5-(4-hydroxyphe-
nyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide 6a (1 mg). The precursor
was dissolved in 0.2 ml of acetone. Five microliters of 2 M sodium hydroxide
was added, and the vial was capped with a septum seal. [11C]methyl iodide,
carried by a stream of helium, was trapped in the above solution cooled in a
dry ice/acetonitrile bath. The reaction was heated in an 808C water bath for
5 min, then quenched with 0.2 ml of HPLC mobile phase consisting of 70%
acetonitrile/0.1 M ammonium formate. The crude reaction product was
purified by reverse-phase HPLC at 10 ml/min. The radioproduct (tR
=
4.9 min) that was separated from the precursor (tR = 2.8 min) was remotely
collected. After concentration to dryness under reduced pressure and heat
(808C), the radiotracer was reconstituted in ethanol (1 ml) and sterile 0.9%
saline (9.0 ml) and passed through a 0.2 mM sterile filter (Millex-FG, Millipore,
Carrigtwohill, Co. Cork, Ireland) into a sterile, pyrogen-free multi-dose vial.
The average non-decay corrected radiochemical yield for [11C]1a was 16 ꢁ 5%
(n ¼ 12). An aliquot (0.1 ml) was assayed for radioactivity, and checked by
analytical HPLC using the mobile phase described above at 3 ml/min. A single
radioactive peak (tR = 2.9 min) corresponding to 1a was observed. The
specific radioactivity at the end-of-synthesis was calculated by relating
radioactivity to the mass associated with the UV absorbance peak of carrier.
An average specific radioactivity at end-of-synthesis (n ¼ 11) of 235 ꢁ 108
GBq/mmol (6360 ꢁ 2914 mCi/mmol) was obtained.
1-(2-bromophenyl)-4-cyano-[11C]5-(4-methoxyphenyl)-N-(piperidin-1-yl)-
1H-pyrazole-3- carboxamide (½11Cꢄ1b)
Radioligand [11C]1b was prepared from 6b with the same procedure described
for [11C]1a. The final radioligand was obtained with average non-decay
corrected radiochemical yield 8.2 ꢁ 1% (n ¼ 4), radiochemical purity greater
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1033
than 99%, and specific activity 196 ꢁ 15 GBq/mmol (5308 ꢁ 419 mCi/mmol) in
4 runs at the end-of-synthesis.
Inhibition binding assay
The in vitro inhibition binding assays of JHU75528, JHU75575, AM281 and
Rimonabant were performed commercially by NovaScreen (Hanover, MD)
under condition similar to that previously published.46 In brief, membranes
from HEK-293 cells expressing the human recombinant cannabinoid receptor
CB1 were incubated with [3H]CP55,940 (Kd=0.6 nM) at a concentration of
0.5 nM in 50 mM Tris-HCl buffer with 5 mM MgCl2, 5 mg/ml BSA and
2.5 mM EDTA at pH 7.4 for 90 min at 308C. The binding reaction was
terminated by rapid vacuum filtration of the assay contents onto presoaked
(0.5% PEI) Whatman GF/C filters. Radioactivity trapped onto the filters was
assessed using liquid scintillation counting. Non-specific binding was defined
as that remaining in the presence of 1 mM HU-210. The assays were done in
duplicate at multiple concentrations of the test compounds. Binding assay
results were analyzed using a 1 site competition models and IC50 curves were
generated based on a sigmoidal dose response with variable slope. Inhibition
binding affinity values (Ki) were calculated using Cheng-Prusoff equation47.
Determination of partition coefficients of [11C]JHU75528 and [11C]JHU75575
700 mCi of the respective radioligand was added to a separatory funnel
containing 1-octanol (20 ml) and 0.02 M phosphate buffer (pH 7.4, 20 ml) pre-
saturated each other. The mixture was shaken mechanically for 3 min and the
layers were separated. The aqueous layer was discarded and 1-octanol (15 ml)
was transferred to a second separatory funnel containing phosphate buffer
(pH 7.4, 15 ml) and the mixture shaken. 1-Octanol (2 ml each) was pipetted
into four test tubes containing phosphate buffer (2 ml each). The test tubes
were stopped, vortexed for 10 min and centrifuged (5 min, 1000 g). The
1-octanol and aqueous phases (1 ml each) were transferred into counting
tubes. The amount of radioactivity in each tube was measured by a g counter
and corrected for decay. The partition coefficient calculated as follows: P =
counts in 1-octanol/counts in phosphate buffer.
Conclusion
In summary, [11C]JHU75528 and [11C]JHU75575, two high affinity CB1
radiotracers and analogs of the selective CB1 antagonist Rimonabant, have
been developed. The experimental lipophilicities of [11C]JHU75528 and
[11C]JHU75575 are in the optimal range for BBB permeation and substantially
lower than those of all previously described PET tracers for CB1 receptor
imaging. The in vitro results with [11C]JHU75528 and [11C]JHU75575 suggest
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1034
H. FAN ET AL.
that these radioligands are promising potential PET tracers for imaging CB1
receptor with high specific and low non-specific binding. This conclusion is
supported by our recent in vivo studies with [11C]JHU7552842. Animal imaging
studies with [11C]JHU75575 are warranted.
Acknowledgements
The authors wish to thank Mr Robert C. Smoot for his assistance. This
research was supported by Department of Radiology of Johns Hopkins
Medical Institutions.
References
1. Gambi F, De Berardis D, Sepede G, Quartesan R, Calcagni E, Salerno RM,
Conti CM, Ferro FM. Int J Immunopathol Pharmacol 2005; 18: 15–19.
2. Zavitsanou K, Garrick T, Huang XF. Prog Neuropsychopharmacol Biol
Psychiatry 2004; 28: 355–360.
3. Ujike H, Morita Y. J Pharmacol Sci 2004; 96: 376–381.
4. Poncelet M, Barnouin MC, Breliere JC, Le Fur G, Soubrie P. Psychopharmacol-
ogy (Berl) 1999; 144: 144–150.
5. Cota D, Marsicano G, Lutz B, Vicennati V, Stalla GK, Pasquali R, Pagotto U.
Int J Obes Relat Metab Disord 2003; 27: 289–301.
6. Vickers SP, Kennett GA. Curr Drug Targets 2005; 6: 215–223.
7. Smith RA, Fathi Z. IDrugs 2005; 8: 53–66.
8. Rinaldi-Carmona M, Barth B, He
Martinez S, Maruani J, Neliat G, Caput D, Ferrara P, Soubrie
J, Le Fur G. FEBS Lett 1994; 350: 240–244.
´
aulme M, Shire D, Calandra B, Congy C,
´
´
P, Claude Breliere
9. Huestis MA, Gorelick DA, Heishman SJ, Preston KL, Nelson RA, Moolchan ET,
Frank RA. Arch Gen Psychiatry 2001; 58: 322–328.
10. Le Foll B, Goldberg SR. J Pharmacol Exp Ther 2005; 312: 875–883.
11. Berding G, Muller-Vahl K, Schneider U, Gielow P, Fitschen J, Stuhrmann M,
Harke H, Buchert R, Donnerstag F, Hofmann M, Knoop BO, Brooks DJ,
Emrich HM, Knapp WH. Biol Psychiatry 2004; 55: 904–915.
12. Howlett AC. Pharmacology of cannabinoid receptors. Annu Rev Pharmacol
Toxicol 1995; 35: 607–634.
13. Howlett AC. Prostaglandins Other Lipid Mediat 2002; 68-69: 619–631.
14. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC,
Herkenham M, Mackie K, Martin BR, Mechoulam R, Pertwee RG. Pharmacol
Rev 2002; 54: 161–202.
15. Timpone JG, Wright DJ, Li N, Egorin MJ, Enama ME, Mayers J, Galetto G.
AIDS Res Hum Retroviruses 1997; 13: 305–315.
16. Hertzog DL. Expert Opini Ther Patents 2004; 14: 1435–1452.
17. Mathews WB, Ravert HT, Musachio JL, Frank RA, Rinaldi-Carmona M, Barth F,
Dannals RF. J Label Compd Radiopharm 1999; 42: 589–596.
18. Mathews WB, Scheffel U, Finley P, Ravert HT, Frank RA, Rinaldi-Carmona M,
Barth F, Dannals RF. Nucl Med Biol 2000; 27: 757–762.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

SYNTHESIS OF [11C]JHU75528 AND [11C]JHU75575
1035
19. Mathews WB, Scheffel U, Rauseo PA, Ravert HT, Frank RA, Ellames GJ,
Herbert JM, Barth F, Rinaldi-Carmona M, Dannals RF. Nucl Med Biol 2002; 29:
671–677.
20. Katoch-Rouse R, Pavlova OA, Caulder T, Hoffman AF, Mukhin AG, Horti AG.
J Med Chem 2003; 46: 642–645.
21. Willis PG, Katoch-Rouse R, Horti AG. J Label Compd Radiopharm 2003; 46:
799–804.
22. Katoch-Rouse R, Horti AG. J Label Compd Radiopharm 2003; 46: 93–98.
23. Charalambous A, Marciniak G, Shiue CY, Dewey SL, Schlyer DJ, Wolf AP,
Makriyannis A. Pharmacol Biochem Behav 1991; 40: 503–507.
24. Gifford AN, Makriyannis A, Volkow ND, Gatley SJ. Chem Phys Lipids 2002;
121: 65–72.
25. Gatley SJ, Gifford AN, Ding Y-S, Volkow ND, Lan R, Liu Q, Makriyannis A.
Development of PET and SPECT Radioligands for Cannabinoid Receptors. In
Drug Discovery Strategies Methods, Makriyannis A, Biegel D (eds). Marcel
Dekker Inc.: New York, 2004; 129–146.
26. Gatley SJ, Lan R, Volkow ND, Pappas N, King P, Wong CT, Gifford AN, Pyatt B,
Dewey SL, Makriyannis A. J Neurochem 1998; 70: 417–423.
27. Berding G, Muller-Vahl K, Schneider U, Gielow P, Fitschen J, Stuhrmann M,
Harke H, Buchert R, Donnerstag F, Hofmann M, Knoop BO, Brooks DJ,
Emrich HM, Knapp WH. Biolog Psychiatry 2004; 55: 904–915.
28. Li Z, Gifford A, Liu Q, Thotapally R, Ding YS, Makriyannis A, Gatley SJ. Nucl
Med Biol 2005; 32: 361–366.
29. Donohue SR, Halldin C, Pike VW. Bioorg Med Chem 2006; 14: 3712–3720.
30. Laruelle M, Slifstein M, Huang Y. Relationships between radiotracer properties and
image quality in molecular imaging of the brain with positron emission tomography.
Molecular Imaging Biol: MIB: Official publ Acad Mol Imaging 2003; 5: 363–375.
31. Katoch-Rouse R, Chefer SI, Pavlova OA, Vaupel DB, Matochik JA, Caulder T,
Hoffman A, Kimes AS, Mukhin AG, Horti AG. IX Symposium on the Medical
Applications of Cyclotrons, Turku, Finland, 2002.
32. Meurer LC, Finke PE, Mills SG, Walsh TF, Toupence RB, Debenham JS, Goulet
MT, Wang J, Tong X, Fong TM, Lao J, Schaeffer MT, Chen J, Shen CP, Sloan
Stribling D, Shearman LP, Strack AM, Van der Ploeg LH. Bioorg Med Chem Lett
2005; 15: 645–651.
33. Meschler JP, Kraichely DM, Wilken GH, Howlett AC. Biochem Pharmacol 2000;
60: 1315–1323.
34. Lange JH, van Stuivenberg HH, Coolen HK, Adolfs TJ, McCreary AC, Keizer
HG, Wals HC, Veerman W, Borst AJ, de Looff W, Verveer PC, Kruse CG. J Med
Chem 2005; 48: 1823–1838.
35. Kubinyi H. The quantitative analysis of structure-activity relationships. In
Burger’s Medicinal Chemistry and Drug Discovery, vol 1, Wolff ME (ed.) (5th
edn). 1995; 497–571.
36. Shawali AS. J Heterocycl Chem 1977; 14: 375–381.
37. Hassaneen HM, Mousa HH, Abed NM, Shawali AS. Heterocycles 1988; 27:
695–706.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr

1036
H. FAN ET AL.
38. Abdel Hamid AO, El Shiaty FH. Phosphorus Sulfur Relat Elem 1988; 39: 45–49.
39. Francis B, Martinez S, Murielle RC. Derives de 4-cyanopyrazole-3-carboxamide,
Leur preparation et leur application en therapeutique. French Patent 2 856
683-A1, 2004.
40. Dessolin M, Guillerez MG, Thieriet N, Guibe F, Loffet A. Tetrahedron Lett 1995;
36: 5741–5744.
41. Eckelman WC, Reba RC, Gibson RE, Rzeszotarski WJ, Vieras F, Mazaitis JK,
Francis B. J Nucl Med 1979; 20: 350–357.
42. Horti AG, Fan H, Kuwabara H, Hilton J, Ravert HT, Holt DP, Alexander M,
Kumar A, Rahmim A, Scheffel U, Wong DF, Dannals RF. J Nucl Med 2006, in
press.
43. Wilson AA, Jin L, Garcia A, DaSilva JN, Houle S. Appl Radiat Isot 2001; 54:
203–208.
44. Zhang MR, Kida T, Noguchi J, Furutsuka K, Maeda J, Suhara T, Suzuki K.
Nucl Med Biol 2003; 5: 513–519.
45. Osman S, Lundkvist C, Pike VW, Halldin C, McCarron JA, Swahn CG, Ginovart N,
Luthra SK, Bench CJ, Grasby PM, Wikstrom H, Barf T, Cliffe IA, Fletcher A,
Farde L. Nucl Med Biol 1996; 5: 627–634.
46. Thomas BF, Gilliam AF, Burch DF, Roche MJ, Seltzman HH. J Pharmacol Exp
Ther 1998; 285: 285–292.
47. Cheng YC, Prusoff WH. Biochem Pharmacol 1973; 22: 3099–3108.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 1021–1036
DOI: 10.1002/jlcr