A high-yield route to synthesize the P-glycoprotein radioligand [ 11C]N-desmethyl-loperamide and its parent radioligand [ 11C]loperamide

Article abstract of DOI:10.1016/j.bmcl.2013.08.024

N-Desmethyl-loperamide and loperamide were synthesized from α,α-diphenyl-γ-butyrolactone and 4-(4-chlorophenyl)-4- hydroxypiperidine in five and four steps with 8% and 16% overall yield, respectively. The amide precursor was synthesized from 4-bromo-2,2- diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypiperidine in 2 steps with 21-57% overall yield. [¹¹C]N-Desmethyl-loperamide and [ ¹¹C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 20-30% and 10-15% radiochemical yields, respectively, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370-740 GBq/μmol specific activity at EOB.

Full text of DOI:10.1016/j.bmcl.2013.08.024

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5259–5263
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A high-yield route to synthesize the P-glycoprotein radioligand
[11C]N-desmethyl-loperamide and its parent radioligand
[11C]loperamide
⇑
Min Wang, Mingzhang Gao, Qi-Huang Zheng
Department of Radiology and Imaging Sciences, Indiana University School of Medicine, 1345 West 16th Street, Room 202, Indianapolis, IN 46202, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Desmethyl-loperamide and loperamide were synthesized from
a,a-diphenyl-c-butyrolactone and
Received 8 July 2013
Revised 26 July 2013
Accepted 5 August 2013
Available online 13 August 2013
4-(4-chlorophenyl)-4-hydroxypiperidine in five and four steps with 8% and 16% overall yield, respec-
tively. The amide precursor was synthesized from 4-bromo-2,2-diphenylbutyronitrile and 4-(4-chloro-
phenyl)-4-hydroxypiperidine in 2 steps with 21–57% overall yield. [11C]N-Desmethyl-loperamide and
[
11C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide
with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with solid-phase extrac-
Keywords:
tion (SPE) in 20–30% and 10–15% radiochemical yields, respectively, based on [11C]CO2 and decay
[
[
11C]N-Desmethyl-loperamide ([11C]dLop)
corrected to end of bombardment (EOB), with 370–740 GBq/
l
mol specific activity at EOB.
11C]Loperamide
Ó 2013 Elsevier Ltd. All rights reserved.
P-glycoprotein (P-gp)
Automation
Positron emission tomography (PET)
Brain imaging
P-Glycoprotein (P-gp) is a cell membrane-associated protein that
transports a variety of drug substrates, and P-gp plays a role in
preventing exogenous or endogenous toxins from entering the cell.1
P-gpis alsoknownasadenosine triphosphate (ATP)-bindingcassette
(ABC) sub-family B member 1 (ABCB1), or multidrug resistance-
associated protein 1 (MRP1), or cluster of differentiation 243
(CD243), encoded by the ABCB1 gene.2 P-gp is highly expressed at
various physiological barriers including blood–brain barrier (BBB),
blood–testis barrier and blood–tumor barrier.3 P-gp overexpression
has been observed in several cancer types and neurodegenerative
disorders such as Alzheimer’s disease, Parkinson’s disease, and
traumatic brain injury.4 P-gp has become an attractive target for
molecular imaging of cancer and brain diseases using biomedical
imaging technique positron emission tomography (PET). Several
PET radioligands have been investigated for their feasibilities to
visualize P-gp at the BBB.5 Latest promising candidates progressing
to human PET studies are (R)-[11C]verapamil and [11C]N-desmeth-
[
11C]dLop is originally developed and characterized at the
National Institute of Mental Health.8–14 The importance of this
compound as a PET brain imaging agent is well recognized, and
broader research investigation to fully explore and validate the util-
ity of [11C]dLop-PET is important. However, the limited commercial
availability, complicated synthetic procedure, and high costs of
starting materials and precursor can present an obstacle to more
widespread evaluation of this intriguing agent. Wishing to study
this compound in our PET center, we decided to make our own
material by following the literature methods.8 The published
H3C CH3
11CH3
H3CO
H3CO
N
OCH3
OCH3
CN
(R)-[11C]Verapamil
yl-loperamide ([11C]dLop), as indicated in Figure 1.6–14
derived from the parent radiotracer [11C]loperamide (Fig. 1). The
parent compound loperamide is a -opioid receptor agonist drug
[
11C]dLop is
H311C
H311C
N
CH3
OH
N
H
OH
l
O
N
Cl
O
N
Cl
originally developed at Janssen Pharmaceutica and used against
diarrhea resulting from gastroenteritis or inflammatory bowel
disease.15–17
[
11C]N-Desmethyl-loperamide ([11C]dLop)
[
11C]Loperamide
Figure 1. Chemical structure of (R)-[11C]verapamil, [11C]N-desmethyl-loperamide
⇑
Corresponding author. Tel.: +1 317 278 4671.
E-mail address: qzheng@iupui.edu (Q.-H. Zheng).
([11C]dLop) and [11C]loperamide.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.024

5260
M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5259–5263
i
synthesis of [11C]dLop has gaps in synthetic detail, and the key step
methylation with CH3I for synthesis of authentic standard N-desm-
ethyl-loperamide (dLop) gave very poor yield (3%) and was difficult
to reproduce in our hands. The overall chemical yield for the syn-
thesis of dLop with 3 steps was <1%. Consequently, we revisited
the reported literature methods15–17 and investigated alternate
presence of Na2CO3 in BuCOMe to obtain loperamide (9) in 38%
yield.
Synthesis of the target tracer [11C]dLop ([11C]7) and its parent
radioligand [11C]loperamide ([11C]9) is indicated in Scheme 3.
The amide precursor 2 or dLop 7 was labeled by [11C]methyl
triflate ([11C]CH3OTf)18,19 through N-[11C]methylation20,21 at 80 °C
under basic condition (newly ground KOH powder) and isolated
by a semi-preparative high performance liquid chromatography
(HPLC) with a C-18 column and a solid-phase extraction (SPE) with
a disposable C-18 Plus Sep-Pak cartridge (a second purification or
isolation process)22–24 to produce the corresponding pure radiola-
beled compound [11C]7 or [11C]9 in 20–30% or 10–15% radiochem-
ical yield, respectively, decay corrected to end of bombardment
(EOB), based on [11C]CO2. In comparison with the results reported
in the literature,8,10 several significant improvements in the radio-
synthesis have been made. [11C]CH3OTf was used as a radiolabeled
precursor, which is a proven methylation reagent with greater
reactivity than commonly used [11C]methyl iodide ([11C]CH3I),25
and thus, the radiochemical yield of [11C]7 was relatively higher.
However, there was no significant difference for the radiochemical
yield of [11C]9 between the use of [11C]CH3OTf and [11C]CH3I,
because it is much more difficult to 11C-methylate the secondary
amide 7 than primary amide 2 using either [11C]CH3OTf or
approaches and modifications that eventually resulted in
high-yield synthesis of
11C]dLop and its parent radioligand
11C]loperamide starting from very beginning materials 4-bromo-
2,2-diphenylbutyronitrile, 4-(4-chlorophenyl)-4-hydroxypiperidine
and -diphenyl- -butyrolactone that was superior to previous
a
[
[
a,a
c
works or addressed more synthetic details to reveal and explain
technical tricks. In this letter we provide complete experiment
procedures, yields, analytical details and new findings for this
high-yield synthetic route, as well as dLop, loperamide and amide
precursor, and present a fully automated radiosynthesis of [11C]dLop
and [11C]loperamide with [11C]methyl triflate ([11C]CH3OTf) in
relatively high radiochemical yields.
As indicated in Scheme 1, the amide precursor 2 was synthe-
sized according to the literature method with modifications. When
we applied the reaction condition reported in the literature8 to
synthesize compound 1 from commercially available 4-bromo-
2,2-diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypi-
peridine in the presence of N,N-diisopropylethylamine (DIPEA) in
CH3CN, the yield was 61%. The reaction condition was optimized
in the presence of NaI and Na2CO3 in CH3CN to afford the desired
product 1 in 70% yield. Hydrolysis of nitrile 1 with pellet KOH in
tBuOH gave the precursor 2 in 35% yield. With the newly ground
KOH powder, the yield was increased to 81% in a shortened
reaction time (from 3 days to 2 days).
[
11C]CH3I. Fortunately, metabolism study indicated that [11C]dLop
is superior to [11C]loperamide in measuring P-gp function at the
BBB.10 It is important to note that newly ground KOH powder
would help the N-[11C]methylation of amide precursor and signif-
icantly increase the radiochemical yield of both [11C]7 and [11C]9.
Meanwhile, small amount of the precursor (0.3–0.5 mg) was used
for radiolabeling instead of large amount of the precursor
(1.0–1.5 mg), which improved the chemical purity of the final
tracer solution. In addition, in order to make more product radioac-
tivity, we also modified the reported semi-preparative HPLC condi-
tions including column, mobile phase and flow rate to shorten the
retention time of [11C]7 and [11C]9. Addition of NaHCO3 to quench
the radiolabeling reaction and to dilute the radiolabeling mixture
prior to the injection onto the semi-preparative HPLC column for
purification gave better separation of [11C]7 from its amide precur-
sor 2 or [11C]9 from its precursor 7.22–24,26 Therefore, the radio-
chemical yields for [11C]7 and [11C]9 in our method (20–30% and
10–15%) are relatively higher than that reported previously (18%
and 11.3% 1.4%).8,10 The radiosynthesis was performed in a
home-built automated multi-purpose 11C-radiosynthesis module,
allowing measurement of specific radioactivity during synthe-
sis.27–29 This 11C-radiosynthesis module includes the overall design
of the reaction, purification and reformulation capabilities of the
prototype system. In addition, 11C-tracer specific activity (SA,
Attempts to methylate the amide 2 with CH3I provided dLop 7
in very low yield (<2%). Alternatively, we turned to another
synthetic route as designed and outlined in Scheme 2. This
synthetic route is based on the reported literature methods15–17
with significant modification and optimization. Ring opening was
accomplished by treatment of
a,a-diphenyl-c-butyrolactone with
HBr in AcOH to afford 4-bromo-2,2-diphenylbutyric acid 3 in 83%
yield. Transformation of acid 3 to its corresponding acid chloride
4 was achieved with SOCl2. Condensation of acid chloride 4 with
aqueous primary amine provided intermediate I, which was rear-
ranged spontaneously under the same reaction condition to form
hydrobromide salt 5 in 60% yield. Neutralization of the hydrobro-
mide salt with 1 N NaOH yielded the free base, and gaseous HCl
passed through the refluxed free base in CHCl3 to afford the ring
opening compound
6 in 68% yield. Treatment of x-chloro-
2,2-diarybutyramide 6 with 4-(4-chlorophenyl)-4-hydroxypiperi-
i
dine in the presence of KI and Na2CO3 in BuCOMe obtained dLop
7 in 25% yield. As shown in Scheme 2, condensation of acid chloride
4 with aqueous secondary amine provided intermediate II. The
intermediate was rearranged spontaneously under the same
reaction condition to form ammonium salt 8 in 50% yield, which
was treated with 4-(4-chlorophenyl)-4-hydroxypiperidine in the
GBq/
uct delivery for compounds purified by the HPLC-portion of the
system.29,30 The SA was in a range of 370–740 GBq/
mol at EOB.
SA is defined as the radioactivity per unit mass of a radionuclide
or a labeled compound. SA (MBq/mg) = 3.13 Â 109/A Â t1/2, where
A is the mass number of the radionuclide, and t1/2 is the half-life
lmol at EOB) can be automatically determined prior to prod-
l
OH
Br
NC
NC
N
OH
DIPEA, CH3CN
or NaI, Na2CO3, CH3CN
HN
+
Cl
Cl
1, 61-70%
OH
O
H2N
C
N
KOH
tBuOH
Cl
2, 35-81%
Scheme 1. Synthesis of amide precursor (2).

M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5259–5263
5261
O
Cl
OH
O
O
O
Br
Br
33% wt HBr
AcOH
SOCl2
CHCl3
4, 99%
3, 83%
HBr
H3CN
O
O
H3CHN
NHCH3
O
C
Cl
Br
40% CH3NH2, Na2CO3
H2O, toluene
1. NaOH, toluene
2. HCl gas
6, 68%
5, 60%
I
OH
HN
O
Br
(H3C)2N
N(CH3)2
O
Cl
Br
KI, Na2CO3, iBuCOMe
40% (CH3)2NH, Na2CO3
H2O, toluene
8, 50%
OH
O
II
H3CHN
C
N
OH
HN
Cl
Cl
N-Desmethyl-loperamide (dLop, 7), 25%
Na2CO3, iBuCOMe
OH
O
(H3C)2N
C
N
Cl
Loperamide (9), 38%
Scheme 2. Synthesis of N-desmethyl-loperamide (dLop, 7) and loperamide (9).
OH
O
OH
O
11CH3OTf, DMSO
KOH, 80 oC, 5 min
H311CHN
C
N
H2N
C
N
Cl
11C]N-Desmethyl-loperamide ([11C]dLop, [11C]7), 20-30%
Cl
2
[
OH
O
OH
O
(H3C)H311CN
C
11CH3OTf, DMSO
KOH, 80 oC, 5 min
N
H3CHN
C
N
Cl
Cl
[
11C]Loperamide ([11C]9), 10-15%
7
Scheme 3. Synthesis of [11C]N-desmethyl-loperamide ([11C]dLop, [11C]7) and [11C]loperamide ([11C]9).
in hours of the radionuclide. For carbon-11, carrier-free 11C, maxi-
mum (theoretical) 11C SA = 340,918 GBq/ mol.31 Actual SAs of the
370 to 925 GBq/
Our study has proved that the maximum in-target 11C SA is
ꢀ925 GBq/ mol (25 Ci/
mol) at EOB in our cyclotron and [11C]CH3-
OTf system. The SA of the title tracer was 370–740 GBq/ mol at
EOB, which was similar to the values previously reported by our
lab. A historical perspective on the SA of radiopharmaceticals indi-
cated it is difficult to achieve high SA for the 11C-tracers.31 How-
ever, a recent paper reported the maximum in-target 11C SA
lmol at EOB according to our previous works.
l
11C-tracers in the PET chemistry facility are depended on two
parts: (1) carrier from the 11C-target, and (2) carrier from the
11C-radiosynthesis unit.30 Furthermore, actual SA for the 11C-trac-
ers synthesized by 11C-methylation with [11C]CH3OTf in our PET
chemistry facility is depended on two parts: (1) carrier from the
cyclotron consisted of the 11C gas irradiation target system, and
(2) carrier from the [11C]CH3OTf system, 11C radiolabeled precursor
or called 11C radiolabeled methylating reagent. The 11C gas target
we used is the Siemens RDS-111 Eclipse cyclotron 11C gas target.
The [11C]CH3OTf production system we used is an Eckert & Ziegler
Modular Lab C-11 Methyl Iodide/Triflate module, convenient gas
phase bromination of [11C]methane and production of [11C]CH3OTf,
a ‘dry’ method using Br2 different with other ‘dry’ method using I2
and ‘wet’ method using LiAlH4 and HI. Our system will have much
less 12C carrier-added in comparison with other ‘dry’ method and
‘wet’ method.19 The SA for our 11C-tracers usually ranges from
l
l
l
2775 GBq/lmol (75Ci/lmol) at EOB has been obtained, using GE
PETtrace cyclotron and TRACERLab FXC automated synthesizer
for the synthesis of a 11C-tracer, [11C]GR103545.32 Chemical purity
and radiochemical purity were determined by analytical HPLC.33
The chemical purity of the precursors and reference standards 2,
7, and 9 was >96%. The radiochemical purity of the target tracers
[
11C]7 and [11C]9 was >99% determined by radio-HPLC through
c
-ray (PIN diode) flow detector, and the chemical purity of [11C]7
and [11C]9 was >90% determined by reverse-phase HPLC through
UV flow detector. A C-18 Plus Sep-Pak cartridge instead of rotatory

5262
M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5259–5263
22. Gao, M.; Wang, M.; Mock, B. H.; Glick-Wilson, B. E.; Yoder, K. K.; Hutchins, G.
D.; Zheng, Q.-H. Appl. Radiat. Isot. 2010, 68, 1079.
23. Wang, M.; Gao, M.; Miller, K. D.; Zheng, Q.-H. Steroids 2011, 76, 1331.
24. Wang, M.; Gao, M.; Miller, K. D.; Sledge, G. W.; Zheng, Q.-H. Bioorg. Med. Chem.
Lett. 2012, 22, 1569.
evaporation was used to significantly improve the chemical purity
of the tracer solution.22–24,33 In this study, the Sep-Pak purification
further increased the chemical purity by 10–20%.22–24
The experimental details and characterization data for com-
pounds 1–9 and for the tracers [11C]7 and [11C]9 are given.34
In summary, a high-yield synthetic route to PET P-gp radioli-
gand [11C]dLop and its parent radioligand [11C]loperamide has
been developed. This new synthetic approach provided amide
precursor, dLop and loperamide in high overall chemical yields.
An automated self-designed multi-purpose [11C]-radiosynthesis
module for the synthesis of [11C]dLop and [11C]loperamide has
been built, featuring the measurement of specific activity by the
on-the-fly technique. The radiosynthesis employed N-[11C]methyl-
ation radiolabeling on nitrogen position of the amide precursor.
Radiolabeling procedures incorporated efficiently with the most
commonly used [11C]methylating agent, [11C]CH3OTf, produced
by gas-phase production of [11C]methyl bromide ([11C]CH3Br) from
our laboratory. The target tracers were isolated and purified by a
semi-preparative HPLC combined with SPE procedure in relatively
high radiochemical yields, short overall synthesis time, and high
specific activity. These results facilitate the potential preclinical
and clinical PET studies of [11C]dLop and [11C]loperamide as brain
P-gp at BBB imaging agents in animals and humans.
25. Allard, M.; Fouquet, E.; James, D.; Szlosek-Pinaudm, M. Curr. Med. Chem. 2008, 15,
235.
26. Gao, M.; Wang, M.; Hutchins, G. D.; Zheng, Q.-H. Appl. Radiat. Isot. 2008, 66, 1891.
27. Mock, B. H.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd. Radiopharm. 2005,
48, S225.
28. Mock, B. H.; Glick-Wilson, B. E.; Zheng, Q.-H.; DeGrado, T. R. J. Labelled Compd.
Radiopharm. 2005, 48, S224.
29. Wang, M.; Gao, M.; Zheng, Q.-H. Appl. Radiat. Isot. 2012, 70, 965.
30. Gao, M.; Wang, M.; Miller, K. D.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 23,
4342.
31. Lapi, S. E.; Welch, M. J. Nucl. Med. Biol. 2013, 40, 314.
32. Nabulsi, N. B.; Zheng, M.-Q.; Ropchan, J.; Labaree, D.; Ding, Y.-S.; Blumberg, L.;
Huang, Y. Nucl. Med. Biol. 2011, 38, 215.
33. Zheng, Q.-H.; Mock, B. H. Biomed. Chromatogr. 2005, 19, 671.
34. (a). General. All commercial reagents and solvents were purchased from
Sigma–Aldrich and Fisher Scientific, and used without further purification.
[
11C]CH3OTf was prepared according to a literature procedure.19 Melting points
were determined on
a MEL-TEMP II capillary tube apparatus and were
uncorrected. 1H NMR and 13C NMR spectra were recorded at 500 and
125 MHz, respectively, on a Bruker Avance II 500 MHz NMR spectrometer
using tetramethylsilane (TMS) as an internal standard. Chemical shift data for
the proton resonances were reported in parts per million (ppm, d scale) relative
to internal standard TMS (d 0.0), and coupling constants (J) were reported in
hertz (Hz). Liquid chromatography-mass spectra (LC–MS) analysis was
performed on an Agilent system, consisting of an 1100 series HPLC
connected to
configured
a
for
diode array detector and
positive-ion/negative-ion
a
1946D mass spectrometer
electrospray ionization.
Acknowledgments
Chromatographic solvent proportions are indicated as volume: volume ratio.
Thin-layer chromatography (TLC) was run using Analtech silica gel GF
uniplates (5 Â 10 cm2). Plates were visualized under UV light. Normal phase
flash column 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. Semi-preparative reverse-phase (RP) HPLC
purification for compounds 7 and 9 employed a Shimadzu LC-20AT pump,
and a SPD-M20A diode array detector set at 254 nm. All moisture- and air-
sensitive reactions were performed under a positive pressure of nitrogen
maintained by a direct line from a nitrogen source. Analytical HPLC was
This work was partially supported by Indiana State Department
of Health (ISDH) Indiana Spinal Cord & Brain Injury Fund (ISDH
EDS-A70-2-079612). 1H and 13C NMR spectra were recorded on a
Bruker Avance II 500 MHz NMR spectrometer in the Department
of Chemistry and Chemical Biology at Indiana University Purdue
University Indianapolis (IUPUI), which is supported by a NSF-MRI
grant CHE-0619254.
performed using a Prodigy (Phenomenex) 5
mobile phase 3:1:1 CH3CN/MeOH/20 mM, pH 6.7 phosphate (buffer solution);
flow rate 1.0 mL/min; and UV (225 nm) and -ray (PIN diode) flow detectors.
lm C-18 column, 4.6 Â 250 mm;
c
Semi-preparative HPLC was performed using a Prodigy (Phenomenex) 5 lm C-
References and notes
18 column, 12 nm, 10 Â 250 mm; mobile phase 60% CH3CN/40% H2O for [11C]7
and 70% CH3CN/30% H2O for [11C]9; 5.0 mL/min flow rate; UV (225 nm) and
c-
ray (PIN diode) flow detectors. C18 Plus Sep-Pak cartridges were obtained from
1. Moerman, L.; Dumolyn, C.; Boon, P.; De Vos, F. Nucl. Med. Biol. 2012, 39, 121.
2. Bartels, A. L. Curr. Pharm. Des. 2011, 17, 2771.
3. Mairinger, S.; Erker, T.; Muller, M.; Langer, O. Curr. Drug Metab. 2011, 12, 774.
4. Pahnke, J.; Wolkenhauer, O.; Krohn, M.; Walker, L. C. Curr. Alzheimer Res. 2008,
5, 396.
5. Wanek, T.; Mairinger, S.; Langer, O. J. Labelled Compd. Radiopharm. 2013, 56, 68.
6. Elsinga, P. H.; Franssen, E. J. F.; Hendrikse, N. H.; Fluks, L.; Weemaes, A. M. A.;
van der Graaf, W. T. A.; DeVries, G. E.; Visser, G. M.; Vaalburg, W. J. Nucl. Med.
1996, 37, 1571.
7. Takano, A.; Kusuhara, H.; Suhara, T.; Ieiri, I.; Morimoto, T.; Lee, Y. J.; Maeda, J.;
Ikoma, Y.; Ito, H.; Suzuki, K.; Sugiyama, Y. J. Nucl. Med. 2006, 47, 1427.
8. Lazarova, N.; Zoghbi, S. S.; Hong, J.; Seneca, N.; Tuan, E.; Gladding, R. L.; Liow, J.-
S.; Taku, A.; Innis, R. B.; Pike, V. W. J. Med. Chem. 2008, 51, 6034.
9. Seneca, N.; Zoghbi, S. S.; Shetty, H. U.; Tuan, E.; Kannan, P.; Taku, A.; Innis, R. B.;
Pike, V. W. Nucl. Med. Biol. 2010, 37, 335.
Waters Corporation (Milford, MA). Sterile Millex-FG 0.2
obtained from Millipore Corporation (Bedford, MA).
lm filter units were
(b). 4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylbutanenitrile (1).
Method A. To a stirred suspension of 4-(4-chlorophenyl)-4-hydroxypiperidine
(705 mg, 3.3 mmol) in CH3CN (5 mL) was added DIPEA (1.16 mL, 6.7 mmol),
followed by 4-bromo-2,2-diphenylbutyronitrile (1.0 g, 3.3 mmol). The reaction
mixture was heated and refluxed overnight. After concentration in vacuo, the
residue was dissolved in CH2Cl2 and purified by column chromatography with
ammonium hydroxide solution (2 M) in MeOH/CH2Cl2 (from 0 to 6:250) to afford
compound
1 (0.88 g, 61%) as a white solid. Method B. A mixture of
4-bromo-2,2-diphenylbutyronitrile (8.0 g, 26.7 mmol), 4-(4-chlorophenyl)-4-
hydroxypiperidine (5.92 g, 28.0 mmol), NaI (4.40 g, 29.4 mmol) and Na2CO3
(5.65 g, 53.3 mmol) in CH3CN (50 mL) was heated and refluxed overnight. The
solvent was removed in vacuo. The residue was dissolved in CH2Cl2 and washed
with water, brine and dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The crude product was purified by column chromatography with
ammonium hydroxide solution (2 M) in MeOH/CH2Cl2 (from 0 to 3:125) to
afford compound 1 (8.05 g, 70%) as a white solid, mp 106–108 °C (lit.8 108–
109 °C). 1H NMR (CDCl3): d 7.43–7.28 (m, 14H), 2.76 (d, J = 11.5 Hz, 2H),
2.66–2.63 (m, 2H), 2.54–2.51 (m, 2H), 2.44 (dt, J = 2.0, 12.0 Hz, 2H), 2.08 (dt,
J = 4.0, 13.0 Hz, 2H), 1.69 (dd, J = 2.0, 14.0 Hz, 2H), 1.61 (br s, 1H).
(c). 4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-2,2-diphenylbutanamide (2).
To a stirred suspension of compound 1 (8.0 g, 18.6 mmol) in tBuOH (100 mL) was
added newly ground KOH powder. The reaction mixture was heated and refluxed
for 2 days. The solvent was removed in vacuo. The residue was dissolved in
CH2Cl2, washed with water, brine and dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The crude product was purified by column
chromatography with ammonium hydroxide solution (2 M) in MeOH/CH2Cl2
(from 1:20 to 3:50) to afford compound 2 (6.76 g, 81%) as a white solid, mp 208–
210 °C (lit.8 208–210 °C). 1H NMR (CDCl3): d 7.41 (d, J = 8.5 °Hz, 2H), 7.32–7.27
(m, 14H), 6.54 (s, 1H), 5.72 (s, 1H), 2.80 (d, J = 10.5 Hz, 2H), 2.66 (t, J = 7.0 Hz, 2H),
2.44–2.35 (m, 4H), 2.08 (t, J = 11.0 Hz, 2H), 1.69 (d, J = 12.5 Hz, 2H), 1.27 (s, 1H).
13C NMR (CDCl3): d 176.8, 146.8, 143.3, 132.9, 128.8, 128.5, 127.2, 126.2, 70.8,
60.0, 55.0, 49.6, 38.2, 35.8. LC-MS (ESI, m/z): Calcd for C27H30ClN2O2 ([M+H]+)
449.2, found: 449.2.
10. Zoghbi, S. S.; Liow, J.-S.; Yasuno, Y.; Hong, J.; Tuan, E.; Lazarova, N.; Gladding, R.
L.; Pike, V. W.; Innis, R. B. J. Nucl. Med. 2008, 49, 649.
11. Liow, J.-S.; Kreisl, W.; Zoghbi, S. S.; Lazarova, N.; Seneca, N.; Gladding, R. L.;
Taku, A.; Herscovitch, P.; Pike, V. W.; Innis, R. B. J. Nucl. Med. 2009, 50, 108.
12. Seneca, N.; Zoghbi, S. S.; Liow, J.-S.; Kreisl, W.; Herscovitch, P.; Jenko, K.;
Gladding, R. L.; Taku, A.; Pike, V. W.; Innis, R. B. J. Nucl. Med. 2009, 50, 807.
13. Kreisl, W. C.; Liow, J.-S.; Kimura, N.; Seneca, N.; Zoghbi, S. S.; Morse, C. L.;
Herscovitch, P.; Pike, V. W.; Innis, R. B. J. Nucl. Med. 2010, 51, 559.
14. Kannan, P.; Brimacombe, K. B.; Kreisl, W. C.; Liow, J.-S.; Zoghbi, S. S.; Telu, S.;
Zhang, Y.; Pike, V. W.; Halldin, C.; Gottesman, M. M.; Innis, R. B.; Hall, M. D.
Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 2593.
15. Stokbroekx, R. A.; Vandenberk, J.; Van Heertum, A. H. M. T.; van Laar, G. M. L.
W.; Van der Aa, M. J. M. C.; Van Bever, W. F. M.; Janssen, P. A. J. J. Med. Chem.
1973, 16, 782.
16. Janssen, P. A. J.; Niemegeers, C. J. E. J.; Stokbroekx, R. A.; Vandenberk, J. US
Patent No. 3714159, 1973.
17. Yaksh, T. L.; Maycock, A. L. US Patent No. 6573282 B1, 2003.
18. Jewett, D. M. Int. J. Radiat. Appl. Instrum. A 1992, 43, 1383.
19. Mock, B. H.; Mulholland, G. K.; Vavrek, M. T. Nucl. Med. Biol. 1999, 26, 467.
20. Wang, M.; Gao, M.; Steele, B. L.; Glick-Wilson, B. E.; Brown-Proctor, C.; Shekhar,
A.; Hutchins, G. D.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2012, 22, 4713.
21. Gao, M.; Shi, Z.; Wang, M.; Zheng, Q.-H. Bioorg. Med. Chem. Lett. 2013, 23, 1953.
(d). 4-Bromo-2,2-diphenylbutyric acid (3).
A mixture of a,a-diphenyl-c-
butyrolactone (20.0 g, 83.9 mmol) in 33% wt HBr in AcOH (60 mL) was stirred

M. Wang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5259–5263
5263
at room temperature for 2 days. The precipitate was collected by filtration,
washed with water and toluene. The crude product was crystallized from iPr2O to
afford compound 3 (22.2 g, 83%) as a white solid, mp 137–138 °C (lit.15 135–
137 °C). 1H NMR (CDCl3): d 7.34–7.27 (m, 10H), 3.10–3.07 (m, 2H), 2.97–2.93 (m,
2H).
to warm to room temperature. The mixture was extracted with CHCl3. The
organic layer was dried over anhydrous Na2SO4, filtered and concentrated in
vacuo. The residue was crystallized from iBuCOMe to afford compound 8 (1.72 g,
50%) as a whitesolid, mp 186–187 °C (lit.15 181–182 °C). 1H NMR (CDCl3): d 7.55–
7.45 (m, 10H), 4.86 (t, J = 6.5 Hz, 2H), 3.83 (s, 3H), 3.47 (t, J = 6.5 Hz, 2H), 2.98 (s,
3H).
(e). 4-Bromo-2,2-diphenylbutanoyl chloride (4). To
a stirred suspension of
compound 3 (10.0 g, 31.4 mmol) in CHCl3 (70 mL) was added SOCl2 dropwise.
The reaction mixture was heated and refluxed for 4 h. The solvent was removed
in vacuo to afford the crude acidchloride4 (10.4 g, 99%)as a yellow oil, whichwas
used for next step without further purification.
(j). 4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-N,N-dimethyl-2,2-diphenyl-
butanamide (loperamide, 9).
A
mixture of 4-(4-chlorophenyl)-4-
hydroxypiperidine (123 mg, 0.58 mmol), Na2CO3 (231 mg, 2.18 mmol) in
iBuCOMe (18 mL) was distilled azeotropically to dry with the aid of a Dean–
Stark trap. After cooling to 80 °C, compound 8 (200 mg, 0.58 mmol) was added.
The reaction mixture was heated and refluxed overnight. The solvent was
removed in vacuo. The residue was diluted with water, and extracted with CHCl3.
The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered
and concentrated in vacuo. The crude product was first purified by column
chromatography with ammonium hydroxide solution (2 M) in MeOH/CH2Cl2
(1:10). Then the product purified by silica gel column was injected onto onto a
(f). N-(Tetrahydro-3,3-diphenyl-2-furylidene)methylamine hydrobromide (5). To a
stirred mixture of 40% aqueous methylamine (1.03 mg, 13.2 mmol) and Na2CO3
(1.24 g, 11.7 mmol) in water (10 mL) and toluene (8 mL) was added compound 4
(3.95 g, 11.7 mmol) in toluene (5 mL) dropwise at 0 °C. The reaction mixture was
allowed to warm to room temperature and the precipitate was collected by
filtration. The solid was dissolved in CHCl3 and dried over anhydrous Na2SO4,
filtered and concentrated in vacuo. The residue was crystallized from iBuCOMe to
afford compound 5 (2.3 g, 60%) as a white solid, mp 158–159 °C (lit.15 159–
161 °C). 1H NMR(CDCl3): d12.7(s, 1H), 7.46–7.20(m, 10H), 4.88(t, J = 6.5 Hz, 2H),
3.28 (d, J = 4.0 Hz, 3H), 3.23 (t, J = 6.5 Hz, 2H).
Luna C18 column (5
lm, 100 A, 10 Â 250 mm), and eluted at 5.0 mL/min 40%
solvent A (water/0.1% TFA) and 60% solvent B (acetonitrile/0.1% TFA) to afford
compound 9 (126 mg, 38%, TFA salt) as a white solid, mp 103–104 °C. 1H NMR
(CDCl3): d 11.10 (s, 1H), 7.43–7.40 (m, 4H), 7.37–7.26 (m, 10H), 5.44 (s, 1H), 3.34
(d, J = 10.0 Hz, 2H), 3.20 (q, J = 10.5 Hz, 2H), 2.94 (s, 3H), 2.76–2.75 (m, 2H), 2.64–
2.61 (m, 2H), 2.44–2.40 (m, 2H), 2.29 (s, 3H), 1.83 (d, J = 14.5 Hz, 2H). 13C NMR
(CDCl3): d 144.7, 138.9 133.6, 129.2, 128.8, 128.0, 127.9, 126.1, 69.1, 60.2, 55.6,
49.2, 39.6, 39.4, 37.4, 35.6. LC–MS (ESI, m/z): Calcd for C29H34ClN2O2 ([M+H]+)
477.2, found: 477.2.
(g). 4-Chloro-N-methyl-2,2-diphenylbutanamide (6).
A cooled solution of
compound 5 (1.8 g, 5.4 mmol) in water (2 mL) was adjusted pH to 12 with 2 N
NaOH. The mixture was extracted with toluene. The organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to
give the free base. The base was dissolved in CHCl3. The mixture was heated and
refluxed while dry HCl gas was bubbled throughfor 1 h. The solventwas removed
in vacuo. The residue was crystallized from iPr2O to afford compound 6 (1.06 g,
68%) as a white solid, 150–151 °C (lit.17 150–152 °C). 1H NMR (CDCl3): d 7.31–
7.21 (m, 10H), 5.42 (s, 1H), 3.52 (t, J = 8.0 Hz, 2H), 2.88 (t, J = 8.0 Hz, 2H), 2.76 (d,
J = 5.0 Hz, 3H).
(k). General procedure for preparation of [11C]N-desmethyl-loperamide ([11C]dLop,
[
11C]7) and [11C]loperamide ([11C]9). [11C]CO2 was produced by the 14N(p, 11C
a)
nuclear reaction in the small volume (9.5 cm3) aluminum gas target provided
with the Siemens RDS-111 Eclipse cyclotron. The target gas consisted of 1%
oxygen in nitrogen purchased as a specialty gas from Praxair, Indianapolis, IN.
(h).
4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-N-methyl-2,2-diphenyl-
butanamide (N-desmethyl-loperamide, 7). A mixture of 4-(4-chlorophenyl)-4-
hydroxypiperidine (350 mg, 1.67 mmol), KI (27.6 mg, 0.17 mmol), Na2CO3
(264 mg, 2.49 mmol) in iBuCOMe (20 mL) was distilled azeotropically to dry
with the aid of a Dean-Stark trap. After cooling to 80 °C, Compound6 (500 mg,
1.66 mmol) was added. The reaction mixture was heated and refluxed overnight.
The solvent was removed in vacuo. The residue was diluted with water, and
extracted with CHCl3. The organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was
first purified by column chromatography with ammonium hydroxide solution
(2 M) in MeOH/CH2Cl2 (1:10). Then the product purified by silica gel column was
Typical irradiations used for the development were 55
30 min on target. The production run produced approximately 45.5 GBq of
11C]CO2 at EOB. In a small reaction vial (5 mL), the precursor 2 or 7 (0.3–0.5 mg)
lA beam current and
[
was dissolved in DMSO (400 lL). To this solution was added the newly ground
KOH powder (1–2 mg). No carrier-added (high specific activity) [11C]CH3OTf that
was produced by the gas-phase production method19 from [11C]CO2 through
[
11C]CH4 and [11C]CH3Br with silver triflate (AgOTf) column was passed into the
reaction vial at room temperature, until radioactivity reached a maximum
(ꢀ2 min), and then the reaction vial was isolated and heated at 80 °C for 5 min.
The contents of the reaction vial were diluted with NaHCO3 (0.1 M, 1 mL), and
injected onto the semi-preparative HPLC column with 3 mL injection loop for
purification. The product fraction was collected in a recovery vial containing
30 mL water. The diluted tracer solution was then passed through a C-18 Sep-Pak
Plus cartridge, and washed with water (5 mL Â 4). The cartridge was eluted with
EtOH (1 mL Â 2), followed by 10 mL saline, to release [11C]7 or [11C]9. The eluted
injected onto a Luna C18 column (5
lm, 100 A, 10 Â 250 mm), and eluted at
5.0 mL/min with 50% solvent A (water/0.1% TFA) and 50% solvent B (acetonitrile/
0.1% TFA) to afford compound 7 (238 mg, 25%, TFA salt) as a pale yellow solid, mp
225–226 °C. 1H NMR (CDCl3): d 11.30 (s, 1H), 7.39–7.28 (m, 10H), 7.16–7.15 (m,
4H), 5.76 (s, 1H), 5.53 (d, J = 5.0 Hz, 1H), 3.37 (d, J = 10.5 Hz, 2H), 3.22 (q,
J = 10.5 Hz, 2H), 3.03–3.02 (m, 2H), 2.88–2.86 (m, 2H), 2.75 (d, J = 4.5 Hz, 3H),
2.46–2.41 (m, 2H), 1.89 (d, J = 14.0 Hz, 2H). 13C NMR (CDCl3): d 175.3, 144.7,
141.9, 133.7, 129.3, 128.9, 128.4, 128.2, 126.1, 69.1, 59.6, 55.6, 49.4, 35.7, 33.6,
27.1. LC–MS (ESI, m/z): Calcd for C28H32ClN2O2 ([M+H]+) 463.1, found: 463.1. The
free base used as precursor was prepared by dissolving the TFA salt in water and
basified with concd ammonium solution, followed by extraction with CHCl3,
dried over anhydrous Na2SO4, filtered and concentrated in vacuo.
product was then sterile-filtered through a sterile vented Millex-GS 0.22 lm
cellulose acetate membrane, and collected into a sterile vial. Total radioactivity
(2.3–4.9 GBq for [11C]7 and 1.2–2.5 GBq for [11C]9) was assayed and total volume
(10–11 mL) was noted for tracer dose dispensing. The overall synthesis,
purification and formulation time was 30–40 min from EOB. Retention times in
the analytical HPLC system were: tR 2 = 3.58 min, tR 7 = 5.11 min, tR 9 = 6.24 min,
tR
preparative HPLC system were: tR 2 = 5.62 min, tR 7 = 8.78 min, tR
[ [
11C]7 = 5.11 min, and tR 11C]9 = 6.24 min. Retention times in the semi-
(i). Dimethyl(tetrahydro-3,3-diphenyl-2-furylidene)ammonium bromide (8). To a
stirred mixture of 40% aqueous dimethylamine (1.35 mg, 12.0 mmol) and
Na2CO3 (2.54 g, 24.0 mmol) in water (8 mL) was added compound 4 (3.38 g,
10.0 mmol) in toluene (5 mL) dropwise at 0 °C. The reaction mixture was allowed
[
11C]7 = 8.78 min; and tR 7 = 5.35 min, tR 9 = 7.65 min, tR 11C]9 = 7.65 min. The
[
radiochemical yields were 20–30% for [11C]7 and 10–15% for [11C]9, respectively,
decay corrected to EOB, based on [11C]CO2.