Imaging evaluation of 5HT2C agonists, [11C]WAY-163909 and [11C]vabicaserin, formed by Pictet-Spengler cyclization

Article abstract of DOI:10.1021/jm401802f

The serotonin subtype 2C (5HT_2C) receptor is an emerging and promising drug target to treat several disorders of the human central nervous system. In this current report, two potent and selective 5HT_2C full agonists, WAY-163909 (2) a

Full text of DOI:10.1021/jm401802f

Article
pubs.acs.org/jmc
Imaging Evaluation of 5HT2C Agonists, [11C]WAY-163909 and
[11C]Vabicaserin, Formed by Pictet−Spengler Cyclization
Ramesh Neelamegam,† Tim Hellenbrand,‡ Frederick A. Schroeder,†,§ Changning Wang,†
and Jacob M. Hooker*,†
†Department of Radiology, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard
Medical School, Charlestown, Massachusetts 02129, United States
‡Department of Pharmacy, Ludwig-Maximilians University, D-81377, Munich, Germany
§Center for Human Genetic Research, Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114, United States
S
* Supporting Information
ABSTRACT: The serotonin subtype 2C (5HT2C) receptor is an emerging and promising
drug target to treat several disorders of the human central nervous system. In this current
report, two potent and selective 5HT2C full agonists, WAY-163909 (2) and vabicaserin (3),
were radiolabeled with carbon-11 via Pictet−Spengler cyclization with [11C]formaldehyde
and used in positron emission tomography (PET) imaging. Reaction conditions were
optimized to exclude the major source of isotope dilution caused by the previously
unknown breakdown of N,N-dimethylformamide (DMF) to formaldehyde at high temperature under mildly acid conditions. In
vivo PET imaging was utilized to evaluate the pharmacokinetics and distribution of the carbon-11 labeled 5HT2C agonists. Both
radiolabeled molecules exhibit high blood−brain barrier (BBB) penetration and nonspecific binding, which was unaltered by
preadministration of the unlabeled agonist. Our work demonstrates that Pictet−Spengler cyclization can be used to label drugs
with carbon-11 to study their pharmacokinetics and for evaluation as PET radiotracers.
positron emission tomography; however, direct carbon-11
incorporation into some of the most promising lead candidates
(certain benzodiazepines) was difficult because no pendent
methyl group or fluorine was available for PET isotope
substitution. Two molecules of particular interest as scaffolds
for ligand development were thus inaccessible because of
limitations in reaction methods for carbon-11 incorporation.
The potent and selective compounds 2 (WAY-163909) and 3
(vabicaserin) are agonists that exhibit high affinity (Ki of ∼10
and 3 nM, respectively) and good efficacy for 5HT2C (EC50 = 8
nM each) and have been characterized extensively in preclinical
drug trials.8,9,22−24
In order to overcome the synthesis challenge of carbon-11
incorporation for 2 and 3, [11C]formaldehyde was used in a
Pictet−Spengler cyclization. Using an in situ formation method
of [11C]formaldehyde from [11C]methyl iodide developed in
our lab,25 we were able to prepare [11C]2 and [11C]3 . In the
process of doing so, we determined that under the acidic
conditions required for product formation, the solvent DMF
degrades to generate formaldehyde, consequently reducing the
specific radioactivity of the product. Through detailed isotope
utilization (deuterium and carbon-13) we pinpointed and
eliminated the sources of formaldehyde while simultaneously
demonstrating that in situ formation of isotopically labeled
formaldehyde is generalizable and can be used to install carbon
INTRODUCTION
■
The serotonin-2C receptor subtype (5HT2C) is an important
G-protein-coupled receptor that has been implicated in many
central nervous system disorders including, among others,
obesity, anxiety, and depression.1−11 5HT2C receptors are
distributed heterogeneously in the mammalian brain with high
densities biased to subcortical regions such as the choroid
plexus. Sufficient densities for imaging exist in the hypothal-
amus, globus pallidus, and substantia nigra. Lower densities of
the receptors have been identified in the cortex and
cerebellum.12−14 Of the diseases 5HT2C has been shown to
cause or contribute to, obesity has been at the forefront of
research, and new drugs targeting 5HT2C (e.g., lorcaserin,
hereafter referred as 1) are the first gaining Food and Drug
Administration (FDA) approval in more than a decade.15,16 To
date, a number of 5HT2C agonists (Figure 1) have been
generated and used in preclinical and clinical studies.8,17 In
addition to a functional role in eating disorders, the receptor
has also been implicated in mood behavior, for example, by
mediating effects of selective serotonin reuptake inhibitors
(SSRIs) and atypical antipsychotics.18−20
Although many studies suggest that dysfunction of 5HT2C
receptors contribute to important brain-related disorders,1−11
direct links between these diseases and 5HT2C receptor
abnormalities have been difficult to elucidate. This is in part
due to the lack of a method for determining 5HT2C receptor
concentration as a function of disease in humans. We have
previously labeled and evaluated arylazepines21 as ligands for
Received: November 21, 2013
Published: February 3, 2014
© 2014 American Chemical Society
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Figure 1. 5HT2C receptor full agonists in preclinical and clinical studies.
a
Scheme 1. Synthesis of WAY-163909 Labeling Precursor (11) and Reference Standard (2)
a
Reagents and conditions: (i) H2 (45 psi), 5% Pd/C, EtOH, rt, 65%; (ii) (a) (S)-2-methoxy-2-phenylacetyl chloride, Et3N, DCM; (b) separation by
column chromatography, 30%; (iii) dilute H2SO4, 70 °C, 8 h, 30%; (iv) 2-methyl-2-oxazoline, pTsOH (cat.), 165 °C, 3 h, 50%; (v) (a) dilute H2SO4,
ο
70 C, 8 h; (b) aq NaOH; (c) 2.0 M Et2O−HCl, rt, 50% (three steps), 97% ee; (vi) 37% aq HCHO, TFA/EtOH, rt, 70%.
a
Scheme 2. Synthesis of Vabicaserin Labeling Precursor (18) and Reference Standard (3)
a
Reagents and conditions: (i) cyclopentadiene, 37% aq HCHO/HCl, EtOH, 5 οC, 10 h; (ii) H2 (45 psi)/10% Pd/C, EtOH/EtOAc (1:1), rt, 10 h,
65%; (iii) (a) ditoluoyl-D-tartaric acid, IPA; (b) separation by crystallization, 30%; (iv) 5% aq NaOH, 90%, 97.5% ee; (v) 2-methyl-2-oxazoline,
pTsOH (cat.), 165 οC, 3 h, 55%; (vi) (a) dilute H2SO4, 70 °C, 8 h; (b) aq NaOH; (c) 2.0 M Et2O−HCl, rt, 50% (three steps), 97% ee; (vii) 37% aq
HCHO, TFA/EtOH, rt, 60%.
and hydrogen isotopes (singly or in concert). These isotopalogs
may themselves warrant preclinical drug evaluation. We further
demonstrate that high specific activity [11C]2 and [11C]3, and
by extension derivatives thereof, can be synthesized. Finally, we
evaluated the potential of [11C]2 and [11C]3 as scaffolds for
PET radiotracer development. Both molecules exhibit high
brain uptake in rats and non-human primates dominated by
nonspecific binding which we are now in the process of tuning
through structure modification.
RESULTS AND DISCUSSION
■
Chemistry. Retrosynthetic analyses of 2 and 3 were possible
provided that carbon-11 could be incorporated at the last step
of the synthesis and provided that the corresponding primary
amines (precursors 11 and 18) could be derived. Schemes 1
and 2 represent our routes to syntheses of precursors 11 and
18, respectively. The development of a synthesis route for
preparing chiral labeling precursors 11 and 18 were more
challenging than expected. For 2 synthesis, neither chiral
crystallization of 6 with (−)-2,3-dibenzoyl-L-tartaric acid and
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Figure 2. Experiments to determine mechanism of specific activity erosion. (A) Synthesis of [*C]2 using in situ generated [*C]CH2O (generated
from [*C]CH3I/oxidant/solvent at 80 °C for 3 min). (B) Aliquots of the reaction mixture injected in the LC−MS instrument (UV, 254 nm). (C)
Taken together, these data shown [12C]CH2O are generated from DMF and TFA at elevated temperatures. DMF should be avoided for high specific
activity.
ditoluyl-D-tartartic acid nor resolution of diastereomers
following reaction of 6 with 1S-(+)-10-camphorsulfonyl
chloride was successful. Reaction of racemic 6 with a chiral
mandelic acid derivative [(R)-(−)-α-methoxyphenylacetic acid]
was ultimately successful. To isolate optically active materials,
racemic compound 6 was treated with an enantiopure (R)-
(−)-α-methoxyphenylacetyl chloride to give two diastereomers
7 and 8. Separation of diastereomer 7 was achieved by column
chromatography, and the structures of 7 and 8 were confirmed
by single crystal X-ray diffraction (XRD) analysis (Figure 1 in
Supporting Information). Removal of the mandelic acid derived
chiral auxiliary cleavage to afford enantio-enriched 11 was not
initially achieved using conditions predicted from the literature
to proceed efficiently (these attempts included 1.0 M LAH in
THF, saturated HCl in methanol at reflux, 5% NaOH in
methanol at reflux, and lithium triethylborohydride (super-
hydride)). Fortunately, the use of dilute sulfuric acid at
moderate temperatures (65 °C) did provide 7 albeit in
moderate yield. More harsh conditions led to decomposition
to unidentified products. Nonetheless, sufficient (multimilli-
gram) quantities of the chiral precursor to 2 (enantiomeric
excess of 97%) were obtained for carbon-11 labeling (Scheme
1).
The synthesis of 3 and its precursor 18 followed the
published patent procedures.26 Resolution of the precursor
enantiomers was achieved by cocrystallization of an early
intermediate using ditoluyl-D-tartartic acid (Scheme 2). Using
this route, we were able to produce 18 for use in carbon-11
radiochemistry and PET imaging. Reference compounds 2 and
3 were synthesized from precursors 11 and 18, respectively,
using a Pictet−Spengler cyclization with formaldehyde under
conditions optimal for isolated yield but not for radiochemical
synthesis.
half-life (t1/2 = 20.4 min). Moreover, we needed to adapt the
cyclization method to be compatible (in a one-pot strategy)
with conditions for in situ generation of [11C]formaldehyde
from [11C]methyl iodide. Initially we determined that 2 could
be formed from 11 at a high temperature (115 °C) in DMF
and trifluoroacetic acid (TFA) using nonradioactive in situ
[12C]CH2O (generated from substoichiometric amounts of
[12C]CH3I with an oxidant, trimethylamine N-oxide (TMAO)).
Reactions with N-methylmorpholine-N-oxide (NMMNO) and
quinuclidine N-oxide (QNO) were successful but provided
lower yield than TMAO. TMAO is also commercially available
and inexpensive.25 To further confirm the labeling position and
compound identity, we used substoichiometric [13C]CH3I and
an excess of TMAO in DMF at 80 °C followed by treatment
with precursor 11, TFA, and H2O at 115 °C to enrich the
expected 50 ppm resonance observed by 13C NMR. Analysis of
the [13C]2 by LC−-MS lead to the observation of an isotope
distribution suggestive of isotopic dilution (i.e., the 99%
enriched [13C]CH3I led to a product on 80−90% enriched).
Dilution of the isotope when applied in the context of carbon-
11 was indeed determined to provide isolated products with
unacceptably low specific radioactivity (0.02 mCi/nmol) for
proper evaluation as a PET radiotracer for 5HT2C receptors.
To determine the source of [12C]CH2O, we systematically
replaced each reaction component starting with the two methyl
sources TMAO and DMF. Previously, we had determined that
TMAO under milder conditions did not decompose to form
formaldehyde, but in order to completely rule out the
contribution from TMAO, we synthesized an oxidant without
methyl groups, QNO. Under the same reaction conditions used
with TMAO and in the presence of only [13C]CH3I, we
observed the formation of [12C]2 by LC−MS (Figure 2C). By
use of perdeuterated N,N-dimethylformamide (DMF-d7) as
solvent, deuterium incorporation was observed by LC−MS
(product m/z, 217; Figure 2 in Supporting Information),
indicating that decomposition of DMF-d7 to formaldehyde-d2
To optimize labeling conditions for carbon-11, we needed to
dramatically reduce the reaction time required for form-
aldehyde condensation and cyclization due to the short carbon
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(CD2O) occurs at high temperatures under mild acidic
conditions. We are unaware of existing data in the literature
describing the formation of formaldehyde from DMF and have
not yet made steps toward elucidating a potential reaction
mechanism, although these steps are planned.
and [11C]3 exhibited high initial blood−brain barrier (BBB)
penetration and good retention in the brain over the scanning
time (60 min) when administered intravenously to Sprague−
Dawley rats (Figure 3). Although whole-brain time−activity
For the present goal unintended formaldehyde formation
was prevented by replacing DMF with N,N-diethylformamide
(DEF). Presumably, DEF can decompose (by analogue) to
acetaldehyde, but we did not observe reaction products by LC−
MS indicative of the subsequent condensation and cyclization
of 11 with acetaldehyde. Satisfyingly, under reaction conditions
with DEF no isotopic dilution was observed using [13C]CH3I
with either TMAO or QNO as the oxidant to generate
[13C]CH2O. Ultimately, TMAO in DEF with TFA proved to
be the most efficient conditions for carbon-11 labeling, and
under these conditions we were able to isolate high specific
radioactivity products.
Radiolabeling of [11C]2 and [11C]3. After optimization,
with a good chemical synthesis method in hand to prevent
isotope dilution while affecting the reaction in reasonable
reaction times, carbon-11 radiolabeling procedures was
performed as follows for imaging: an amount of 4 mg of
TMAO was dissolved in DEF and cooled to −10 °C in a
reactor, and [11C]CH3I was trapped. Then the mixture was
heated to 80 °C for 3 min. In a separate vial 1.2 0.3 mg of the
alkylamines, 11/18, was dissolved in a mixture of DEF, water,
and TFA. This solution was then transferred into the reaction
vial, and heating was continued at 115 °C for 6 min (Scheme
3). To quench the reaction, 1 mL of 0.1% formic acid was
Figure 3. Top: Summed PET images (0−60 min) following injection
of [11C]2: baseline scan (0.265 mCi); image acquired following
pretreatment (5 min prior) with 2 (1 mg/kg, 0.303 mCi). Images are
dose corrected. Bottom: Summed PET images (0−60 min) following
injection of [11C]3: baseline scan (0.96 mCi); image acquired
following pretreatment (5 min prior) with 3 (1 mg/kg, 0.99 mCi).
Images are dose corrected. Whole-brain time−activity curves were
generated from imaging data.
Scheme 3. Radiosynthesis of [11C]2 and [11C]3 Using in Situ
[11C]CH2O (Generated from [11C]CH3I/Me3N+O−/DEF at
a
80 °C for 3 min)
curves generated from imaging data are presented, heteroge-
neous binding was observed for both labeled compounds. In
order to determine the sensitivity of uptake to pretreatment
(and potential saturation of the 5HT2C receptor sites), rodents
were pretreated in separate experiments with the corresponding
unlabeled ligand (e.g., 3, 1 mg/kg iv, was administered 5 min
prior to [11C]3 imaging). Pharmacokinetics of [11C]2 were
altered by drug treatment; pharmacokinetics of [11C]3 were
not. The more rapid washout of [11C]2 can be attributed to
several phenomena including receptor saturation, changes in
arterial plasma concentration, or changes in metabolism. We
were encouraged by the data to further evaluate specificity
(saturability) of [11C]2 binding and thus moved to non-human
primates where we have extensive experience performing MR-
PET imaging with arterial-derived and metabolite corrected
blood data.21,27
a
RCY is an isolated, formulated, and decay corrected yield.
added, and the entire reaction mixture was purified by reversed-
phase semipreparative HPLC. The final product was reformu-
lated into 10% EtOH with saline using C-18 solid-phase
exchange (SPE). The chemical and radiochemical purity of the
final products, [11C]2 and [11C]3, were tested by analytical
HPLC. The identities of the respective products were
confirmed by analytical HPLC with additional co-injection of
corresponding reference standards (Figures 3 and 4 in
Supporting Information). The average time required for the
synthesis from end of cyclotron bombardment to end of
synthesis and purification was 50 min [11C]2 and [11C]3. The
Evaluation of [11C]2 in Papio anubis Baboon. Prior to
non-human primate imaging, we experimentally determined
that the partition coefficient (log D) between pH 7.4 phosphate
buffered saline (PBS) and octanol was 1.53 0.13 (n = 3). In
addition, the predisposition of [11C]2 to bind plasma protein
(ppb) was determined with a baboon plasma sample after 10
min of incubation time at room temperature. Relatively high
average radiochemical yield was [11C]2 (1.5
0.5%; decay-
corrected to trapped [11C]CH3I; n = 3) and [11C]3 (1.3
0.2%; decay-corrected to trapped [11C]CH3I; n = 3). Chemical
and radiochemical purities were >97% in all instances. Specific
activities of [11C]2 and [11C]3 were 0.85 0.2 mCi/nmol (n =
3) and 1.18 0.20 mCi/nmol (n = 3), respectively, at the end
of synthesis (EOS).
(26.5
0.30%) amounts of [11C]2 were free at equilibrium
which was encouraging given previously reported correlations
between plasma protein binding and nonspecific brain
homogenate binding.28,29
Preliminary Imaging Evaluation of [11C]2 and [11C]3.
Using a small animal PET-CT, we determined that both [11C]2
To further investigate the brain permeability of [11C]2 and
specific binding to 5HT2C receptors, we administered the
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Figure 4. (A) Summed PET images (0−80 min) following injection of [11C]2: (left) baseline scan (2.08 mCi); (middle) image acquired following
pretreatment (10 min prior) with 2 (1 mg/kg, 2.33 mCi). Images are dose corrected. (B) Whole-brain time−activity curves generated from PET
imaging data. Maximum uptake is observed at 20 min after injection. Neither a reduction in uptake nor a change in the distribution or kinetics was
observed between the baseline and blocking curves.
labeled compound in a paired baseline/pretreatment protocol
to Papio anubis baboon. As in rodents, [11C]2 exhibited very
good BBB penetration and high brain uptake over the scanning
time (80 min) when the [11C]2 (2.08 mCi) was administered
intravenously (Figure 4A). Unlike in rodents, pretreatment
with 2 (1 mg/kg, 2.33 mCi) 10 min prior to injection of [11C]2
did not alter the pharmacokinetics of radioactivity in the whole
brain. Time−activity curves generated from PET imaging data
provided that maximal uptake occurs at 20 min after injection.
Neither a reduction in uptake nor a change in the distribution
or kinetics was observed between the baseline and blocking
dose curves (Figure 4B). Arterial blood samples indicated that
there was no change in blood half-life or in compound
metabolism. The differences in TACs for rodents and NHPs
(as well as other species) are not fully understood but also not
uncommonly observed. Current understanding of the ex-
pression of 5HT2C receptors in rat and non-human primate
brain indicates an overall similarity, with differences noted in
subregions including the neocortex, hippocampal subfields, and
substantia nigra,30,31 and range in density from ∼50 to 150
fmol/mg protein.32,33 Little is described about 5HT2C receptors
outside the brain. We noticed in rat a robust uptake of [11C]2
in regions outside the brain but within the skull (Figure 3 top,
baseline, blue). This accumulation may comprise olfactory
epithelium or lacrimal glands, two components modulated by
serotonin. Pretreatment with unlabeled 2 decreased binding in
these peripheral target sites, resulting in altered pharmacoki-
netics of the tracer. This was evidenced by a vertical shift in
early time points of the time−activity curve, to a large extent in
rat (Figure 3 top) and less so in baboon (Figure 4). Further
studies are warranted to quantify species differences in non-
CNS 5HT2C receptors and understand their role in 5-HT
signaling within the brain. Combined, these data suggest that
the signal from [11C]2 and/or its metabolites in the non-human
primate brain is nonspecific and not an indicator of 5HT2C
distribution and density. Nonetheless, given the favorable
properties of low PPB, good metabolic stability, appropriate
lipophilicity, and high BBB penetration, 2 is a good scaffold for
further derivation.
important 5HT2C receptor agonists in both rat and baboon and
have provided insight into CNS pharmacokinetics. Both [11C]2
and [11C]3 exhibit rapid and high brain uptake but with high
nonspecific binding (not necessarily a negative feature for use
of these compounds as preclinical pharmacology tools). Our
radiolabeling strategy is adaptable, and future work will be well
positioned to radiolabel additional 5HT2C drugs or any tracers
to investigate the serotonergic system and others in using PET
imaging.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures, characterization data, radiochemistry, and
acquisition details for in vivo imaging studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone: 617-726-6596. Fax: 617-726-7422. E-mail:
hooker@nmr.mgh.harvard.edu. Address: Athinoula A. Martinos
Center for Biomedical Imaging, Building 149, 13th Street, Suite
2301, Charlestown, MA 02129.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was carried out at the Athinoula A. Martinos
Center for Biomedical Imaging at the Massachusetts General
Hospital, using resources provided by the Center for Functional
Neuroimaging Technologies, P41EB015896, a P41 Regional
Resource supported by the National Institute of Biomedical
Imaging and Bioengineering (NIBIB), National Institutes of
Health. This project was funded by a grant from the National
Institutes of Health (Grant 1R21MH093874). This work also
involved the use of instrumentation supported by the NIH
Shared Instrumentation Grant Program and/or High-End
Instrumentation Grant Program, specifically, Grants
S10RR017208, S10RR026666, S10RR022976, S10RR019933,
and S10RR029495. We thank members of the Hooker research
laboratory for helpful discussions. The authors are grateful to
Grae Arabasz, Shirley Hsu, Joseph Mandeville, and Helen Deng
for assistance during NHP imaging and to Christian Moseley
and Stephen Carlin for technical assistance in the radio-
chemistry laboratory. The authors also thank Prof. Ritter’s
research group, Department of Chemistry, Harvard University,
MA, for single crystal X-ray data collection.
CONCLUSIONS
■
We have developed a one-pot, two-step synthesis of [11C]2 and
[11C]3 by using a Pictet−Spengler type cyclization with in situ
generated high specific activity [11C]formaldehyde. We isolated
sources of isotopic dilution and have provided a general set of
reaction conditions for labeling certain benzodiazepines
isotopically (both stable and unstable) at benzylic position.
We accomplished the first PET imaging evaluation of two
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ABBREVIATIONS USED
■
PET-CT, positron emission tomography−computated tomog-
raphy; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; DEF, N,N-diethylformamide; THF, tetrahydrofuran;
DMF-d7, N,N-dimethylformamide-d7; LAH, lithium aluminum
hydride; TMAO, trimethylamine N-oxide; QNO, quinuclidine
N-oxide; TFA, trifluoroacetic acid; PBS, phosphate buffered
saline; FDA, Food and Drug Administration; NHP, non-human
primate; TAC, time−activity curve; BBB, blood−brain barrier;
PPB, plasma protein binding; XRD, X-ray diffraction
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