[11C]MK-4232: The first positron emission tomography tracer for the calcitonin gene-related peptide receptor

Article abstract of DOI:10.1021/ml400199p

Rational modification of the potent calcitonin gene-related peptide (CGRP) receptor antagonist MK-3207 led to a series of analogues with enhanced CNS penetrance and a convenient chemical handle for introduction of a radiolabel. A number of ¹¹C-

Full text of DOI:10.1021/ml400199p

Letter
pubs.acs.org/acsmedchemlett
[11C]MK-4232: The First Positron Emission Tomography Tracer for the
Calcitonin Gene-Related Peptide Receptor
Ian M. Bell,*,† Steven N. Gallicchio,† Craig A. Stump,† Joseph G. Bruno,∥ Hong Fan,§ Liza T. Gantert,§
Eric D. Hostetler,§ Amanda L. Kemmerer,∥ Melody McWherter,† Eric L. Moore,‡ Scott D. Mosser,∥
Mona L. Purcell,§ Kerry Riffel,§ Christopher A. Salvatore,‡ Sandra Sanabria-Bohorquez,§
́
Donnette D. Staas,† Rebecca B. White,# Mangay Williams,§ C. Blair Zartman,† Jacquelynn J. Cook,§
Richard J. Hargreaves,⊥ Stefanie A. Kane,‡ Samuel L. Graham,† and Harold G. Selnick†
Departments of †Medicinal Chemistry, ‡Pain & Migraine, §Imaging, ∥In Vitro Pharmacology, ⊥Neuroscience & Ophthalmology, and
#Pharmacokinetics Pharmacodynamics & Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486, United
States
S
* Supporting Information
ABSTRACT: Rational modification of the potent calcitonin
gene-related peptide (CGRP) receptor antagonist MK-3207 led
to a series of analogues with enhanced CNS penetrance and a
convenient chemical handle for introduction of a radiolabel. A
number of 11C-tracers were synthesized and evaluated in vivo,
leading to the identification of [11C]8 ([11C]MK-4232), the first
positron emission tomography tracer for the CGRP receptor.
KEYWORDS: MK-4232, calcitonin gene-related peptide receptor, positron emission tomography
MK-3207 (2), and BI-44370, which have all shown clinical
igraine is a common neurovascular disorder, charac-
Mterized by attacks of headache that are often efficacy as acute antimigraine therapies.4−7 Although the clinical
efficacy observed with these novel agents is not in dispute, the
precise site of action of the compounds remains a topic for
debate. It is certainly possible that a CGRP receptor antagonist
(CGRP-RA) could achieve some antimigraine efficacy by
blocking CGRP-induced activation of trigeminovascular pain
pathways in the periphery, but the presence of CGRP and its
receptor in parts of the brain that may be involved in migraine
attacks suggests that a central site of action is also plausible.8
At clinically efficacious doses, both telcagepant (1)9 and MK-
3207 (2)10 (Chart 1) have been found to effectively block the
CGRP receptor in the periphery, based on results from the
accompanied by such symptoms as nausea, photophobia, and
phonophobia.1 It is a leading cause of disability and poses a
significant economic burden.1 Although the precise mecha-
nisms underlying migraine pathogenesis are not fully under-
stood, two theories have been most influential. The vascular
theory attributes migraine headache to dilation of cerebral or
meningeal arteries, while the neural theory holds that migraine
is primarily a disorder of the central nervous system (CNS).2
The 37 amino acid neuropeptide calcitonin gene-related
peptide (CGRP) is thought to play a key role in migraine
pathogenesis.3 It is distributed widely in the central and
peripheral nervous system, and it is known to be a highly
potent vasodilator.3 More recently, significant evidence has
pointed to an important role for CGRP as a neuromodulator in
the brain.2 The CGRP receptor (CGRP-R) is a G protein-
coupled receptor (GPCR) and is heterodimeric in nature,
consisting of the calcitonin receptor-like receptor (CLR) and
receptor activity modifying protein 1 (RAMP1). The
components of the CGRP receptor have been detected in the
smooth muscle layer of intracranial blood vessels, as well as in
the neurons in the trigeminal ganglion.2
Chart 1. Selected CGRP Receptor Antagonists
While a number of key experiments suggested that blocking
CGRP function could have utility for migraine treatment,
definitive evidence has been provided by clinical studies with
the CGRP receptor antagonists olcegepant, telcagepant (1),
Received: May 22, 2013
Accepted: July 10, 2013
Published: July 18, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
capsaicin-induced dermal vasodilation pharmacodynamic
model.11,12 Nonetheless, other evidence, including the
observation that dosing of telcagepant to rhesus monkeys led
to measurable drug levels in their cerebrospinal fluid (CSF), has
led to the suggestion that engagement of central CGRP
receptors might be necessary for maximal antimigraine
efficacy.2
suggesting that there may be significant free drug in the CNS.12
However, it should be noted that the CSF levels of P-gp
substrates like 1 and 2 are unreliable indicators of free brain
concentrations.16
Compound 3 (Chart 2), in which the piperazinone
spirocyclopentyl moiety in 2 was replaced with geminal
In order to directly address the question of central CGRP
receptor occupancy by antagonists such as telcagepant, we
sought to identify a positron emission tomography (PET)
tracer for the CGRP receptor that would be suitable for clinical
use. Such a molecule should possess good membrane
permeability and lack susceptibility for being actively trans-
ported out of the CNS, for example, by the P-glycoprotein (P-
gp) efflux pump. Additionally, an ideal PET ligand should
possess a good binding potential.13 In terms of compound
design, this translates to a good level of receptor affinity relative
to the receptor abundance in the target tissue (Bmax/Kd > 10),
and a moderate lipophilicity to mitigate nonspecific binding
(logD7.0 < 3.5).14
Chart 2. Potential PET Tracers and Precursors
Overall, it seemed that MK-3207 (2) might be a reasonable
starting point for this effort. While 2 was a substrate for the P-
gp transporter (P-gp = 25, Table 1), it had acceptable passive
Table 1. Data for CGRP Receptor Antagonists
ab
,
c
d
e
compd CGRP Ki (pM)
P-gp
monkey fu (%)
CSF:PL (%)
1
770 70 (13)
24
25
33
20
8.9
2.8
9.3
1.7
14
3.8
1.3
2
21 6 (14)
34 11 (27)
110 (2)
9.6
2.9
3
42.9
33.3
20.5
11.7
10.4
4.3
4.35
ND
ND
4.79
6.61
3.12
ND
ND
ND
0.53
ND
0.99
2.64
4
5
22 1 (15)
56 20 (5)
40 12 (8)
46 15 (7)
17 (2)
6
7
8
9
ND
0.2
f
f
f
10
11
12
13
14
15
500 (2)
NM
1.8
dimethyl substituents, retained excellent CGRP-R affinity and
a high P-gp transport ratio. This modification also conferred a
4-fold increase in monkey plasma fu (42.9% for 3 vs 9.6% for 2;
Table 1). In order to reduce the P-gp transport manifested in
compounds 2 and 3, we sought to explore modifications that
would either add steric bulk close to polar functionality or
replace polar groups with lipophilic alternatives.
45 (2)
6.1
94 21 (5)
21 (2)
NM
2.0
0.2
5.1
110 22 (6)
19 7 (4)
NM
1.5
0.8
3.1
a
Mean value standard deviation, where appropriate; the number of
b
replicates is in parentheses. The Ki value for inhibition of 125I-hCGRP
The addition of a benzylic methyl substituent to the
piperazinone ring in 3 produced compound 4 (Chart 2),
which had reduced CGRP receptor affinity but also had slightly
attenuated susceptibility for P-gp transport (Table 1). Similarly,
the addition of a methyl group to the piperazinone nitrogen of
3 afforded compound 5, which also appeared to have a reduced
level of P-gp transport (Table 1). Gratifyingly, combining these
observations led to the tetramethylpiperazinone 6, a potent
CGRP-RA (Ki = 56 pM) with low susceptibility for active
transport by P-gp (transport ratio = 2.8).
The reduction in P-gp transport achieved in modifying 3 (P-
gp = 33) to give 6 (P-gp = 2.8) translated to a relatively modest
increase in CSF levels relative to unbound plasma levels. For 3,
10% of unbound drug was observed in CSF; whereas for 6, 40%
of unbound drug was observed in CSF (Table 1). These results
suggested that 6 represented an improvement over 3 in terms
of CNS penetrance, but that it was not optimal.
binding was determined using membranes from HEK293 cells stably
c
expressing human CLR/RAMP1.11 Human P-gp transport ratio BA/
d
AB determined using LLC-MDR1 cells. Unbound fraction
e
determined in plasma using equilibrium dialysis methodology. Ratio
of CSF AUC to plasma AUC determined in cisterna magna-ported
f
rhesus monkey.12 Accurate values could not be determined due to the
physical properties of compound.
permeability in LLC-PK1 cells (Papp = 28 × 10−6 cm/s) and
lipophilicity (logD7.0 = 2.35), and excellent receptor affinity (Ki
= 21 pM). Since the highest level of the CGRP-R in the brain
of rhesus monkeys was found to be in the cerebellum (Bmax
=
24 nM),15 and using Ki as a surrogate for Kd, the binding
potential for a compound with this level of receptor affinity was
impressive (Bmax/Ki > 1000).
Both 1 and 2 were found to exhibit measurable levels in CSF
following dosing in cisterna magna-ported rhesus monkeys
(Table 1). For each compound, about one-third of the free
plasma concentration was observed in CSF (for 2, monkey
plasma fu = 9.6% and monkey CSF/plasma = 2.9%; Table 1),
Application of the same strategy to 2 led to compounds 7
and 8 (Chart 2). Both 7 and 8 maintained high affinity for the
CGRP-R (Table 1) but, while 7 was a moderate substrate for P-
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ACS Medicinal Chemistry Letters
Letter
gp (P-gp = 9.3), 8 exhibited minimal susceptibility for this
transporter (P-gp = 1.7). Moreover, compound 8 exhibited
CSF levels that corresponded to more than 70% of unbound
plasma levels, indicating an increased level of CNS penetrance
with respect to 2. The corresponding aza-analogue 9, an N-
methylated version of the previously reported MK-8825,17
exhibited significantly increased P-gp susceptibility (P-gp = 14),
perhaps because of the introduction of an additional hydrogen
bond acceptor.
Importantly, the N-methyl substituent on the piperazinone
ring of compounds like 6 and 8 offered a convenient approach
to the synthesis of potential PET ligands: methylation of a
suitable precursor (4 or 7) with [11C]methyl triflate. Indeed,
treatment of 7 with [11C]methyl triflate in acetone followed by
HPLC purification afforded [11C]8, and the other potential
PET tracers shown in Chart 2 were synthesized analogously.
Preliminary evaluations of the uptake kinetics of tracers [11C]
5, [11C]6, and [11C]8 were conducted in anesthetized rhesus
monkeys. Figure 1 illustrates the time-activity curves for [11C]5
and [11C]8 for both cerebellum (circles) and white matter
(triangles).
were intermediate between [11C]5 and [11C]8 (Table 2). Thus,
the magnitude of the apparent specific signal for these tracers
correlated inversely with their susceptibility for P-gp transport
(Table 2).
In addition to increasing steric bulk around the piperazinone
ring in order to attenuate P-gp transport, replacement of the
polar central amide moiety with lipophilic groups was
evaluated. These efforts led to the alkane, alkene, and alkyne
analogues 10−15 (Chart 2), which were prepared as previously
described.18 In general, these compounds maintained excellent
affinity for the CGRP-R, although replacement of the amide
moiety with an ethylene spacer did lead to some reduction in
receptor affinity (compare 10 with 8 and 11 with 9; Table 1).
As expected, these amide replacements conferred increased
lipophilicity, and the indanyl analogues 10, 12, and 14 had
logD7.0 > 4, outside of the target range (Table 2). The high
lipophilicity of compounds 10, 12, and 14 correlated with low
unbound fraction in monkey plasma. Moreover, reliable values
for permeability and P-gp transport could not be determined
for these three compounds, apparently because their poor
physical properties led to high levels of nonspecific binding. For
the corresponding azaindanyl analogues (11, 13, and 15), the
logD7.0 values were more reasonable and the compounds
exhibited good permeability and low susceptibility for P-gp
transport in vitro (Table 2). Additionally, the CSF concen-
trations of 12, 14, and 15 following dosing in rhesus monkeys
approximated the corresponding levels of unbound drug in
plasma (Table 1), indicating that these compounds were indeed
CNS penetrant. Overall, it appeared that this strategy of amide
replacement generally afforded compounds with low P-gp
susceptibility and good CNS penetrance.
On the basis of the overall profiles of compounds 10−15, the
[11C]-tracers were prepared from the corresponding des-methyl
analogues, using essentially identical methodology to that used
to synthesize [11C]8, and uptake kinetics were evaluated in
anesthetized rhesus monkeys. The results of these studies are
summarized in Table 2 in terms of SUV at 80 min postdose in
cerebellum and white matter. On the basis of the ratio of
SUVcerebellum/SUVWM in these preliminary studies, it appeared
that the highest specific signals were obtained with [11C]11,
[11C]13, and [11C]15; intermediate results were observed with
[11C]12 and [11C]14; and a low specific signal was found for
[11C]10. This rank-ordering of the tracers in terms of the
magnitude of their specific signals correlates inversely with their
CGRP Ki values (Table 2), consistent with the use of binding
potential (Bmax/Kd) as a guide to specific receptor binding.
Overall, [11C]8 (MK-4232) had the best in vivo profile of the
tested PET tracers and it was selected for further evaluation.
In radioligand binding assays, compound 8 had excellent
affinity for CGRP receptors from both human (Ki = 46 pM)
and rhesus monkey (Ki = 33 pM). In a cell-based functional
Figure 1. Decay-corrected time−activity curves showing SUV in
cerebellum (circles) and white matter (triangles) for [11C]5 (A) and
[11C]8 (B) in rhesus monkey.
assay, 8 was a potent CGRP receptor antagonist (cAMP IC50
=
Consistent with earlier autoradiography mapping studies, the
highest level of binding activity in the brain was generally
observed in the cerebellum.12 The signal in white matter
appeared to be indicative of nonspecific binding, and the ratio
of the standardized uptake value (SUV) in cerebellum to SUV
in white matter provided an estimate of the specific signal for
the tracer. For [11C]5, the apparent specific signal was modest
(Figure 1A) and the uptake at 80 min postdose in cerebellum
0.097 nM) and its activity was right-shifted about 4-fold by the
addition of 50% human serum (cAMP + HS IC50 = 0.39 nM).
In terms of selectivity, 8 was >30,000-fold selective for the
CGRP receptor versus a standard panel of more than 160
enzymes, receptors, and transporters. Thus, 8 is a highly potent
and selective antagonist of primate CGRP receptors.
Baseline PET scans (Figure 1B) showed good brain uptake
of [11C]8 in rhesus monkeys, with a good specific signal for
CGRP receptors in the cerebellum. The summed baseline PET
images (Figure 2A) revealed a regional distribution consistent
with the known distribution of CGRP receptors in rhesus
was only slightly higher than that in white matter (SUVcerebellum
/
SUVWM ≈ 1.4; Table 2). In contrast, [11C]8 provided a
significantly higher signal in cerebellum than in white matter
(SUVcerebellum/SUVWM ≈ 2.0). The results obtained with [11C]6
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ACS Medicinal Chemistry Letters
Letter
Table 2. Data for Potential PET Tracers
f
approximate SUV @ 80 min
ab
,
c
d
e
g
compd
CGRP Ki (pM)
logD7.0
P-gp
Papp (10−6cm/s)
cerebellum
white matter
SUVcerebellum/ SUVWM
5
22 1 (4)
56 20 (5)
46 15 (7)
500 (2)
ND
ND
3.39
4.33
3.16
4.15
3.36
4.11
3.41
8.9
2.8
1.7
27
29
25
0.7
1.2
1.7
0.8
1.0
1.0
1.2
1.1
0.5
0.7
0.9
0.6
0.6
0.7
0.7
0.7
0.7
1.4
1.7
2.0
1.3
1.7
1.4
1.7
1.6
1.9
6
8
h
h
h
h
10
11
12
13
14
15
NM
1.8
NM
45 (2)
23
h
94 21 (5)
21 (2)
NM
2.0
NM
21
h
110 22 (6)
NM
NM
19 7 (4)
1.5
17
1.3
a
b
Mean value standard deviation, where appropriate; the number of replicates is in parentheses. The Ki value for inhibition of 125I-hCGRP binding
was determined using membranes from HEK293 cells stably expressing human CLR/RAMP1.11 HPLC logD at pH 7.0. Human P-gp transport
c
d
e
f
ratio BA/AB determined at 5 μM test compound using LLC-MDR1 cells. Permeability determined using control LLC-PK1 cells. Specific uptake
g
value observed in cerebellum or white matter of anesthetized rhesus monkeys 80 min after i.v. dosing of the [11C]-tracer. Ratio of specific uptake
h
values in cerebellum and white matter at 80 min postdose. Accurate values could not be determined due to the physical properties of compound.
a
Scheme 1. Synthesis of [11C]8
Figure 2. Overlay of MRI and summed [11C]8 PET images (50−120
min) in rhesus monkey brain: (A) baseline data; (B) data obtained in
the presence of ∼10 μM plasma levels of MK-3207.
monkey brain, and a notably robust signal in the cerebellum.
The images obtained in the presence of ∼10 μM MK-3207
(Figure 2B) illustrate a blockade of binding to the CGRP
receptors in all regions. Taken together, these results
demonstrate that [11C]8 should have utility for the study of
a
Conditions: (a) (1) Di-tert-butyl dicarbonate, CH2Cl2, 0 °C; (2) Di-
central CGRP receptor occupancy.
tert-butyl dicarbonate, DMAP, 80 °C, 96%. (b) Potassium tert-
The methodology used to synthesize compounds 3−15 has
butoxide, THF, −78 °C, 52%. (c) LAH, THF, −78 °C, 78%. (d)
been described.18,19 The synthesis of [11C]8 is outlined in
Methyl 1-aminocyclopentanecarboxylate, NaCNBH3, AcOH, MeOH,
60%. (e) HCl, EtOAc, 0 °C, 99%. (f) AcOH, xylenes, 80 °C, 90%. (g)
Scheme 1. Following protection of [1-(3,5-difluorophenyl)-
ethyl]amine (16) with di-tert-butyl dicarbonate, the bis-Boc-
ChiralPak AD, EtOH/hexane/Et2NH (60:40:0.1), (R)-20 is the
protected amine was allowed to rearrange under basic
conditions to afford the protected amino acid 17. Treatment
of ester 17 with LAH at low temperature provided aldehyde 18,
and reductive amination with methyl 1-amino-cyclopentane-
carboxylate hydrochloride led to amine 19. Removal of the Boc
protecting group from 19 followed by heating in the presence
of acetic acid afforded the racemic piperazinone 20, which was
resolved by chiral HPLC. Alkylation of (R)-20 with ethyl-
bromoacetate and subsequent saponification provided carbox-
second major peak. (h) NaH, ethyl bromoacetate, THF, 0 °C, 87%.
(i) LiOH, H2O, THF, 95%. (j) (R)-5-Amino-1,3-dihydrospiro-
[indene-2,3′-pyrrolo[2,3-b]pyridin]-2′(1′H)-one,20 HATU, NMM,
DMF, 68%. (k) [11C]MeOTf, acetone, 32%.
ylic acid 21. Standard amide coupling of 21 with (R)-5-amino-
1,3-dihydrospiro[indene-2,3′-pyrrolo[2,3-b]pyridin]-2′-(1′H)-
one19,20 gave 7. Lastly, treatment of 7 with [11C]methyl triflate
gave the PET tracer [11C]8.
866
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ACS Medicinal Chemistry Letters
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trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]-
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Stump, C. A.; Zartman, C. B.; Fay, J. F.; Li, C.-C.; Lynch, J. L.; Moore,
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In conclusion, the identification of the first PET tracer for the
CGRP receptor has been described. Compound 8 (MK-4232)
is a highly potent, CNS-penetrant CGRP receptor antagonist.
The PET tracer [11C]8 exhibited good rhesus monkey brain
uptake in vivo and specific binding to central CGRP receptors.
Further characterization of this tracer and its utility for
investigating CGRP receptor occupancy in clinical studies will
be reported elsewhere.
ASSOCIATED CONTENT
* Supporting Information
■
S
Representative experimental procedures and NMR, MS, and
HPLC data for all new test compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(I.M.B.) Phone: +1-215-652-6455. Fax: +1-215-652-7310. E-
mail: ian_bell@merck.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Chris Burgey, Mark Fraley, and Jerome Hochman for
helpful discussions; Aniket Joshi for preparing the images used
in Figures 1 and 2; James Small for 2D NMR analysis of
compound 8; the West Point MS and NMR groups for
spectroscopic data; Mandy Dancho and Maria S. Michener for
dosing and sample collection in cisterna magna-ported rhesus
monkeys; Emily Adarayan, Richard Gundersdorf, Jim Monahan,
Yang Xu, and Rena Zhang for pharmacokinetic analysis; Susan
Crathern and Nicole Pudvah for generating P-gp data; and
Matthew Zrada for plasma protein binding determinations.
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Graham, S. L.; Selnick, H. G.; Vacca, J. P.; Kane, S. A. Pharmacological
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pyrrolo[2,3-b]pyridin]-5-yl]acetamide (MK-3207), a potent and orally
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H.; Salvatore, C.; Hargreaves, R.; Kane, S.; Graham, S.; Sur, C.; Cook,
J. J.; Hostetler, E. [C-11]MK-4232: The First PET Tracer for the
Calcitonin Gene-Related Peptide (CGRP) Receptor and its Evaluation
in Rhesus Monkey, Sixth Annual World Molecular Imaging Congress,
Montreal, Canada, September 23−26, 2009; poster presentation
number 0813.
ABBREVIATIONS
■
CGRP, calcitonin gene-related peptide; CGRP-R, calcitonin
gene-related peptide receptor; CGRP-RA, calcitonin gene-
related peptide receptor antagonist; CLR, calcitonin receptor-
like receptor; CNS, central nervous system; CSF, cerebrospinal
fluid; GPCR, G-protein coupled receptor; HPLC, high-
performance liquid chromatography; LAH, lithium aluminum
hydride; ND, not determined; PET, positron emission
tomography; P-gp, P-glycoprotein; RAMP1, receptor activity
modifying protein 1; SUV, standardized uptake value
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