Synthesis and characterization of [N-methyl-3H]loperamide

Article abstract of DOI:10.1002/jlcr.3198

Loperamide is a piperidine butyramide mu-opiate receptor agonist and currently employed to treat diarrhea. Because a single past report of tritiating loperamide was limited to only a very low specific activity product without technical details or extensive analysis, the synthesis of [N-methyl- ³H]loperamide at high specific activity is now described in detail. An imine precursor was alkylated with [³H]methyl iodide to obtain a quaternary intermediate, which was then reacted with 4-(4-chlorophenyl)-4- hydroxypiperidine to afford the desired product [N-methyl-³H] loperamide, characterized by thin layer chromatography (TLC), HPLC, MS, UV, and proton-decoupled tritium NMR.

Full text of DOI:10.1002/jlcr.3198

Protocols and Methods
Received 12 January 2014,
Revised 23 February 2014,
Accepted 15 March 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3198
Synthesis and characterization of
3
[N-methyl- H]loperamide
*
Crist N. Filer, Judith A. Egan, and Richard P. Nugent
Loperamide is a piperidine butyramide mu-opiate receptor agonist and currently employed to treat diarrhea. Because a
single past report of tritiating loperamide was limited to only a very low specific activity product without technical
details or extensive analysis, the synthesis of [N-methyl-3H]loperamide at high specific activity is now described in detail.
An imine precursor was alkylated with [3H]methyl iodide to obtain a quaternary intermediate, which was then reacted
with 4-(4-chlorophenyl)-4-hydroxypiperidine to afford the desired product [N-methyl-3H]loperamide, characterized by
thin layer chromatography (TLC), HPLC, MS, UV, and proton-decoupled tritium NMR.
Keywords: loperamide; mu-opiate receptor; tritium; tritium NMR
synthesis and complete characterization of high specific
Introduction
activity [N-methyl-3H]loperamide.
Any discussion of the mu-opiate receptor system is unders-
tandably dominated by the topic of analgesia. However, there
are other profound and useful biological effects mediated by this
Results and discussion
crucial binding site besides pain relief. In 1973, Janssen medicinal
chemists described1 the remarkable antidiarrheal properties of
In selecting a strategy to tritium label 1, we initially explored the
method of halogenation–catalytic tritium dehalogenation.
However, for reasons that are not clear, we had no success in
either the bromination or iodination of 1 and it appears that
the literature also contains no examples of this derivatization.
In view of these results, there appeared to be no better tritiation
option than the original Janssen synthesis1 itself, but some
experimental modifications were made to accommodate the
small scale and radiochemistry. Higher boiling solvents were
chosen because the two-step synthesis was conducted in a
sealed glass apparatus. Imine 2 was prepared as reported1 with
nothing unusual noted beyond what was experimentally
described. It was then reacted with high specific activity [3H]
methyl iodide to give the iminium salt 3 as seen in Scheme 1.
Because of instability concerns, this intermediate was not
purified or characterized but instead immediately reacted with
commercially available 4-(4-chlorophenyl)-4-hydroxypiperidine.
After the final reaction, TLC analysis of the crude reaction
mixture revealed significant amounts of desired product
[N-methyl-3H]loperamide (4), which was conveniently purified
by reverse phase HPLC. The observed HPLC purification recovery
was somewhat lower than expected and this may be due in part
to a degree of crude product decomposition during concentration
prior to HPLC injection. The product specific activity was easily
the mu-opiate receptor agonist loperamide (1), based on its
selective activity at the myenteric plexus of the large intestine.
Additionally, the structure of loperamide had been cleverly crafted
(with p-chlorine substitution on the 4-phenylpiperidine) to deprive
it of any appreciable central nervous system (CNS) activity. Over the
years, the life-saving benefit of this valuable substance, especially in
severely dehydrated children, has been well-documented.2
However, beyond its role as an effective medicine, there has been
recent heightened interest in loperamide for an entirely different
reason. The permeability-glycoprotein (P-gp) efflux pump, also
known as the multidrug resistance protein 1 is widely expressed in
various tissues. As its name implies, P-gp readily transports many
drugs across cell membranes, often interfering with their biological
target destination. It has been suggested that some disorders may
be associated with abnormalities of P-gp. For instance, P-gp may
play a role in limiting the access of chemotherapeutic agents to
tumor cytoplasm.3 Furthermore, it has also been proposed that
reduced function of P-gp at the blood–brain barrier may hinder
the clearance of beta-amyloid peptide from the brain, thereby
increasing the chance of amyloid plaque formation and resulting
Alzheimer’s disease.4 Recently, loperamide has been demonstrated
to be a superior substrate for P-gp and may be one of the best
probes to measure its function.5
The tritiation of loperamide has been reported but just briefly
referenced by only a structural reaction scheme lacking any
experimental details.6 Also, the product obtained was of
extremely low (45 mCi/mmol) specific activity, analyzed only by
TLC and without proof of tritium location. Because of the recent
renewed interest in loperamide and our continued fascination
with the opiate receptor family,7 we now describe the
PerkinElmer Life Sciences & Technology Inc., 940 Winter Street, Waltham, MA
02451, USA
*Correspondence to: C. N. Filer, PerkinElmer Life Sciences & Technology Inc., 549
Albany Street, Boston, MA 02118, USA.
E-mail: crist.filer@perkinelmer.com
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.

C. N. Filer et al.
conformation of 1 in solution. The literature appears to contain only
a single study9 of the proton NMR of 1 in solution. Examining the
host–guest inclusion complexation of 1 with beta-cyclodextrin,
these authors also assigned the very same methyl chemical shift
values (δ 2.33 and 2.95 ppm), which we observed for 4 but made
no attempt to explain the dramatic differences for them. A possible
explanation is that because of restricted rotation about the crowded
amide bond, the two amide methyl resonances are a consequence
of their different exposures to the shielding and deshielding regions
of the amide carbonyl and/or phenyl rings. Finally, with striking
evidence that both methyls of 4 are almost equally radiolabelled,
the tritium NMR also demonstrates that mechanistically, there is
apparently no stereochemical preference for either the methylation
of precursor 2 or the ring opening rearrangement of intermediate 3
to product 4.
We have performed the robust reaction sequence reported here a
number of times, and under the described experimental conditions,
it affords tens of mCi of pure product 4 at high specific activity.
Scheme 1. Synthesis of [N-methyl-3H]loperamide (4). Reagents and conditions:
(a) [3H]methyl iodide, methyl ethyl ketone, 65 0C, 8 h. (b) 4-(4-chlorophenyl)-4-
hydroxypiperidine, sodium carbonate, benzene, 65 °C, 1 h.
Experimental
General
determined by mass spectrometry with the appearance of a large
parent ion for 4 at m/z 483. Alternatively, the specific activity could
also be measured by a companion radioassay and UV (ethanol)
mass determination of 4 for which Є260 = 644 for 1.
All chemicals used were reagent grade. Evaporations were carried out on
a Buchi rotary evaporator (model RE 111) at bath temperatures less than
40 °C. Analytical TLC was performed on Analtech silica gel glass plates.
Analytical HPLC was accomplished on a PerkinElmer instrument, and
peak detection was performed simultaneously by UV and an IN/US
Systems Beta RAM Model 3 radioactivity detector. Solution assays were
performed with a PerkinElmer Tri-Carb 3100TR instrument. The mass
spectrum was recorded on a Kratos Model MS25 RF instrument, and
the UV spectrum was performed on a PerkinElmer Lambda 35 UV
spectrophotometer. The proton-decoupled tritium NMR was obtained
on a Bruker 300-MHz instrument with chemical shifts being reported as
parts per million (ppm) downfield from internal tetramethylsilane.
Perhaps the most intriguing spectral property of product 4 was its
proton-decoupled tritium NMR as shown in Figure 1. The two large
singlets observed are attributed to fully tritiated N-methyl groups,
but each of them is also accompanied by a downfield singlet
corresponding to smaller amounts of their C3H2H methyl
isotopologues. Interestingly, the tritium resonances of the two main
radiolabelled methyl peaks are widely separated by virtue of what
apparently are their very dissimilar chemical and magnetic
environments. Recently, the crystal structure of loperamide
monohydrate was measured with high precision by single crystal
X-ray analysis8 and is the same three-dimensional structure as
portrayed in 1. However, this may not necessarily be the actual
[N-Methyl-3H]loperamide (4)
To a solution of 21 (12.6 mg, 0.05 mmol) in 0.25 mL of methyl ethyl ketone
was added 21 Ci (0.25 mmol) of [3H]methyl iodide, and the reaction was
heated at 65 °C for 8 h. in a sealed glass vessel fitted with several break seal
joints. After this time, the vessel was cooled, opened, and 16 mg
(0.076 mmol) of 4-(4-chlorophenyl)-4-hydroxypiperidine (Aldrich C66056),
16 mg of sodium carbonate, and 0.25 ml of benzene were added. The
reaction vessel was then resealed and heated at 65 °C for 1 h. It was then
cooled and opened, and labile tritium was removed by the vacuum
evaporation of several ethanol portions to give 1.1 Ci of crude product. TLC
analysis of the reaction product mixture (silica gel (chloroform: methanol–
ammonium hydroxide (19:1:0.1)) indicated that approximately 60% of it
was desired product 4. Half of the crude product was purified by preparative
reverse phase HPLC (acetonitrile–1% aqueous triethylammonium acetate
(pH 4) (60:40)) to afford 62 mCi (an extrapolated 3% yield based on precursor
2) of product 4, which was 98% radiochemically pure on TLC and HPLC
(same systems as previously mentioned) and also co-chromatographed with
authentic 1 (Aldrich L4762). The specific activity of 4 was measured to be
84.5 Ci/mmol by mass spectrometry with a prominent parent ion peak at
483 m/z. Its UV (ethanol) spectrum was superimposable on that of authentic
1 as well. A proton-decoupled tritium NMR (CD3OD) of 4 is shown in Figure 1
and reveals that the tritium had been exclusively and almost equally installed
on two methyl groups of 4.
Acknowledgement
We would like to acknowledge the contribution of Dr. Puliyer
Srinivasan of PerkinElmer Life Sciences
obtaining the tritium NMR spectrum.
& Technology in
Figure 1. Proton decoupled tritium NMR (CD3OD) of [N-methyl-3H]loperamide.
www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014

C. N. Filer et al.
[3] M. M. Gottesman, T. Fojo, S. E. Bates, Nat. Rev. Cancer 2002, 2,
48–58.
Conflict of Interest
[4] S. Vogelgesang, I. Cascorbi, E. Schroeder, J. Pahnke, H. K. Kroemer,
W. Siegmund, C. Kunert-Keil, L. C. Walker, R. W. Warzok,
Pharmacogenetics 2002, 12, 535–541.
The authors did not report any conflict of interest.
[5] J.-S. Taur, E. L. Schuck, N. Y. Wong, Drug Metab. Lett. 2012, 6, 285–291.
[6] J. Heykants, M. Michiels, A. Knaeps, J. Brugmans, Arzneim.-Forsch.
1974, 24, 1649–1653.
[7] C. N. Filer, J. Labelled Compd. Radiopharm. 2013, 56, 639–648.
[8] J. P. Jasinski, C. J. Guild, A. S. Dayananda, H. S. Yathirajan, A. R. Ramesha,
Acta Cryst. 2012, E68, o539–o540.
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J. Label Compd. Radiopharm 2014
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